|Year : 2013 | Volume
| Issue : 1 | Page : 24-30
|Virulence factors in clinical and commensal isolates of Enterococcus species
Praharaj Ira, Sistla Sujatha, Parija Subhash Chandra
Department of Microbiology, Jawaharlal Institute of Postgraduate Medical Education and Research, Puducherry, India
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|Date of Web Publication||6-Aug-2013|
| Abstract|| |
Background: Enterococci have emerged as important nosocomial pathogens and have been found to possess many virulence factors, some of which are considered very important in the pathogenesis of diseases caused by them. The following study was carried out to evaluate some of the virulence determinants elaborated by strains of enterococci in our setup and to ascertain if these strains differ considerably from commensal strains of enterococci in the expression of these virulence determinants. Materials and Methods: One hundred and fifty-seven isolates of Enterococcus species from clinical specimens were evaluated for the presence of virulence determinants like hemolysin production, gelatinase production and biofilm formation by phenotypic tests. The presence of enterococcal surface protein (esp) gene in the isolates was detected using polymerase chain reaction (PCR). Thirty strains of Enterococcus isolated from fecal samples of patients admitted to the hospital were also tested for the presence of these virulence factors. Strains of Enterococcus from clinical specimens and those present as commensals were compared with respect to the elaboration of virulence factors using Fisher's exact test. Results: The association between biofilm formation and presence of the "esp" gene was not found to be statistically significant. Among the virulence determinants studied, gelatinase production and the "esp" gene were found to be significantly more common in clinical isolates than commensal strains of Enterococcus species. Conclusion: Among the virulence factors, gelatinase and the "esp" gene were more common in clinical isolates than commensal strains. The association between biofilm formation and the presence of "esp" gene was not found to be statistically significant.
Keywords: Biofilms, Enterococcus, hemolysin, gelatinase, polymerase chain reaction, virulence
|How to cite this article:|
Ira P, Sujatha S, Chandra PS. Virulence factors in clinical and commensal isolates of Enterococcus species. Indian J Pathol Microbiol 2013;56:24-30
|How to cite this URL:|
Ira P, Sujatha S, Chandra PS. Virulence factors in clinical and commensal isolates of Enterococcus species. Indian J Pathol Microbiol [serial online] 2013 [cited 2020 Apr 9];56:24-30. Available from: http://www.ijpmonline.org/text.asp?2013/56/1/24/116144
| Introduction|| |
Enterococcus species have been for long considered relatively innocuous organisms, existing as normal commensals of the gastrointestinal tract. But in the last few decades, the number of serious infections caused by these organisms has been steadily increasing.
Apart from the trend of increased drug resistance seen in these organisms (which itself can be considered a virulence factor), quite a few virulence factors elaborated by these organisms have been studied in recent times. Some of these virulence factors, like hemolysin, gelatinase and the ability to form biofilms (enterococcal surface protein), are well studied, whereas other putative virulence factors, like hyaluronidase, are still not conclusively linked with the disease-causing capability of enterococcus strains.
This study was carried out to study the elaboration of virulence factors by strains of Enterococcus isolated from clinical specimens and to determine if there is any significant difference in the virulence factors found in clinical isolates and those in isolates from stool samples. This study also attempted to determine if there is any significant difference in the virulence factors elaborated by vancomycin-resistant strains of Enterococcus species compared to sensitive strains.
| Materials and Methods|| |
The study was conducted in the department of microbiology, Jawaharlal Institute of Postgraduate Medical Education and Research, Pondicherry, from November 2008 to October 2009. A total of 157 clinical isolates of Enterococcus species were included in this study. The samples from which these isolates were obtained included sterile body fluids like blood, cerebrospinal fluid (CSF), peritoneal fluid, pleural fluid, isolates from urine samples and pus and wound swabs. Routine bacteriological methods were followed for the isolation and identification of Enterococcus species. , Thirty isolates of Enterococcus species from stool samples were collected for comparing the virulence factors elaborated by strains isolated from clinical samples and those found as commensals in stool. Bile-esculin azide agar (Hi-Media laboratories, Mumbai, India) was used for the isolation of Enterococcus species from stool specimens. The study was approved by the institutional ethics committee.
Production of hemolysin was determined by plating the enterococcal isolates onto brain heart infusion (BHI) agar supplemented with 5% human blood. Plates were incubated at 37°C and observed after 24 and 48 hours. A clear zone of β hemolysis around the bacterial colonies indicated the production of hemolysin. All strains inoculated onto human blood agar were also inoculated onto agar containing 5% sheep blood. This was to look for strains of Enterococcus, which show β hemolytic colonies on both sheep blood agar and human blood agar. [Figure 1] shows beta-hemolytic colonies of Enterococcus on human blood agar.
|Figure 1: β hemolyti c colonies of a strain of Enterococcus on human blood agar Cytolysin/Hemolysin producti on)|
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Detection of gelatinase production
Enterococcus isolates were inoculated onto peptone yeast extract agar containing gelatin (30 g/L) and incubated at 37°C for 24 hours.  The plates with growth were then cooled to ambient temperature for two hours. Appearance of a turbid halo or zone around the colonies was considered to be an indication of gelatinase production.
Biofilm production was tested for by using three methods - the microtiter plate method, the tube method and the Congo red agar method. Of these three methods, the microtiter plate method and the tube method have been used to study the capability to produce biofilms by enterococci. Congo red agar has been used to test the biofilm producing capability of coagulase negative staphylococci. This medium was included in this study to see if it correlated with the findings by other methods.
Microtiter plate method
This procedure was performed as described earlier with a few modifications. , Colonies of enterococci, which had grown overnight on blood agar were inoculated in trypticase soy broth (TSB) (Hi-media laboratories, Mumbai, India) with 2% sucrose and incubated at 37°C overnight. This overnight growth was diluted 1: 100 in the TSB with sucrose. 200 μL of this diluted inoculum was added onto sterile flat-bottomed polystyrene microtiter plates (Laxbro, Pune, India). The microtiter plates were incubated aerobically at 37°C for 48 hours. At the end of 48 hours, the culture was discarded from the wells. The wells were gently washed with PBS (pH 7.2) to remove nonadherent planktonic cells. Manual washing was done using a multichannel micropipette. The adherent biofilms were then fixed by using 2% sodium acetate for 20 minutes. The plates were then dried at room temperature and finally stained with 0.1% safranin for 20 minutes. The plates were then washed five times as previously described and dried. The absorbance of the biofilm on the bottom surface of each well was determined at 490 nm with an ELISA microplate reader. All strains were inoculated in triplicate on the microtiter plate and all experiments were repeated three times [Figure 2]. For calculating the absorbance, the average of the absorbance for each of the three wells inoculated with a single strain was calculated. All experiments also included three blank wells (i.e., culture medium without any bacteria).
|Figure 2: Biofi lm detecti on in Enterococcus species by microti ter plate assay|
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At 490 nm, biofilm formation was considered to be high when the absorbance was >0.2 OD, moderate when it was between 0.20 and 0.10 and weak/absent when <0.10. 
Tube method for biofilm detection
The method described by Christensen et al. was used to assess biofilm formation.  TSB with 2% sucrose was used in this method. TSB with sucrose was inoculated with a loopful of the microorganisms from an overnight culture and incubated for 24 h at 37°C. The tubes were then decanted and washed with PBS pH 7.2 to remove any non-adherent cells. The tubes were then dried and stained with 0.1% crystal violet for 30 minutes. The excess stain was washed off with distilled water and the tubes were left to dry in inverted position and observed for biofilm formation. A visible film lining the sides and the bottom of the tube was considered to be indicative of biofilm formation. Ring formation at the liquid interface only was not indicative of biofilm formation. Amount of biofilm was scored as 0 - absent, 1 - weak, 2 - moderate or 3 - strong [Figure 3].
Congo red agar method for detecting biofilms
The use of this agar for detecting biofilms was first studied in coagulase negative staphylococci by Freeman et al. A specially prepared medium known as Congo Red Agar (CRA) is used for this test. For preparing, sucrose and Congo red stain were added to BHI agar at a concentration of 50 g/L and 0.8 g/L, respectively.  The Enterococcus strains were inoculated onto CRA and incubated at 37°C for 24 hours. Readings were taken after 24 hours and again after 48 hours. A positive result was indicated by black colonies with black crystalline morphology. Non-slime producers mostly produced pink- or red-colored colonies [Figure 4] and [Figure 5].
|Figure 5: Colony appearance of biofi lm producers and biofi lm nonproducers on Congo Red Agar|
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Esp gene detection by polymerase chain reaction (PCR)
Enterococcal surface protein (esp) sequence-specific primers were used to detect the presence of "esp" gene. The primer sequences chosen were from Shanker et al. 
"esp" forward primer
5'-TTG CTA ATG CTA GTC CAC GAC C-3'
"esp" reverse primer
5'-GCG TCA ACA CTT GCA TTG CCG AA-3'
Colony PCR was optimized for detecting the "esp" gene. 2-3 Enterococcus colonies from an overnight growth on 5% blood agar were suspended with a sterile microtip in the PCR reaction mixture. Prior to optimizing the colony PCR, alkaline lysis method followed by phenol chloroform extraction was tried for DNA extraction. However, since colony PCR gave comparable results as the more cumbersome alkaline lysis method, it was eventually followed. For the colony PCR, the reaction mixture contained 12.5 μl of master mix (Red Dye Master Mix, Bangalore Genei), 8.5 μl of Millipore water, 2 μL (5 pmols) each of the forward and reverse primers (Sigma Aldrich) and the bacterial colonies.
The following PCR conditions were used:
94°C for 2 min for the first cycle (initial denaturation)
For the next 30 cycles
94°C for 1 min for denaturation
54°C for 1 min for annealing
72°C for 1 min for extension
For the last cycle 72°C for 7 min for the final extension.
PCR products were subjected to electrophoresis in a 1% agarose gel using 1% TBE buffer and documented using a gel documentation system [Figure 6].
Comparison of virulence factors elaborated by clinical isolates and isolates from stool.
Thirty strains of Enterococcus isolated from stool samples (colonizers) were tested by the above-mentioned methods for detection of hemolysin, gelatinase and biofilm production.
Comparison between the virulence factors expressed by clinical isolates and isolates from stool carriers was done using Fisher's exact test. Comparison between the virulence factor expression by vancomycin-resistant enterococci and vancomycin-sensitive enterococci was also done using Fisher's exact test.
| Results|| |
One hundred and fifty-seven clinical isolates and 30 Enterococcus isolates from stool were tested for the presence of virulence factors.
Virulence factors in Enterococcus isolates from clinical samples
Hemolysin production in enterococci
Of the 157 samples tested for the presence of hemolysin, 21% showed beta-hemolysis on human blood agar [Table 1]. 36.7% of the urine isolates tested showed beta-hemolysis on human blood agar, whereas only 15% of the blood and exudates isolates tested were positive for hemolysin.
Gelatinase production in enterococci
Gelatinase production was seen in only 19% of the clinical isolates tested. 20.9% of the urine isolates tested showed gelatinase activity while 31% of the isolates from exudates and blood samples were positive for gelatinase activity.
Biofilm production by isolates of enterococci
Detection of biofilm by different methods
Three methods of detecting biofilm production were tried in our study [Table 2]. The microtiter plate method was the method with which the other two methods (Tube method and Congo Red Agar method) were compared. Among the clinical isolates tested for biofilm production, 53% were found to be positive by microtiter plate assay. 56% of the enterococcal isolates from urine were found to be positive for biofilm formation by the microtiter plate assay.
Sensitivity and Specificity of biofilm detecting methods
Congo Red Agar
The sensitivity and specificity of the Congo red agar method was found to be 30% and 77% when compared with the microtiter plate method for detecting biofilms [Table 3].
The sensitivity and specificity of the tube method was found to be 61% and 68% as compared to the microtiter plate method [Table 3].
Presence of "esp" gene
PCR for detecting the presence of "esp" gene was carried out on 58 clinical isolates. The colony PCR method was found to give comparable results to the PCR done on DNA extracted by the rapid alkaline lysis method. Of the 58 clinical isolates, 32 were vancomycin resistant Enterococcus (VRE) isolates. Forty-five clinical isolates were found to possess the "esp" gene. Of the 28 isolates from urine, 22 (78.6%) were found to have the "esp" gene. Among the VRE isolates, 28 isolates (87.5%) were positive for the "esp" gene.
Correlation between biofilm formation and presence of "esp" gene
Of the 58 clinical isolates tested for the presence of "esp" gene, only 28 were found to be both "esp"-positive and biofilm producers by the microtiter plate method. Of the 13 'esp"- negative strains, seven were found to be biofilm producers while six were negative for biofilm production.
There was no statistically significant difference in the proportion of biofilm producers among "esp"-positive and "esp"- negative strains (P value = 0.7490). Strains having the "esp" gene are not more likely to produce biofilms than strains without the gene.
Correlation between virulence factors and vancomycin resistance
Hemolysin production in VRE and VSE strains
Four out of the 32 isolates of VRE showed beta hemolysis on human blood agar whereas 29 of the 96 isolates of vancomycin sensitive Enterococcus (VSE) tested for hemolysin production were positive. The difference in the expression of hemolysin by VRE strains and VSE strains was not found to be significant using Fisher's exact test (P = 0.2296).
Gelatinase production in VRE and VSE strains
13 out of the 32 VRE strains and 20 out of the 105 VSE isolates were positive for gelatinase production. There was no significant difference in the elaboration of gelatinase by VRE and VSE strains (P = 0.0750).
Biofilm production in VRE and VSE strains
13 out of the 32 VRE strains and 70 out of the 125 VSE isolates were biofilm producers. No significant difference was observed in the proportion of VRE isolates producing biofilms and the proportion of VSE isolates with biofilm production (P0 = 0.1644 by Fisher's exact test)
Presence of "esp" gene in VRE and VSE strains
A majority of the VRE strains were found to be positive for the "esp" gene (28 out of 32 VRE isolates). However, a considerable proportion of the VSE strains tested were also positive for the "esp" gene (17 out of the 26 strains tested). The difference between these two groups was statistically not significant (P = 0.0605). Both VRE and VSE strains were equally likely to possess the "esp" gene.
Elaboration of virulence factors by Enterococcus isolates from stool
A total of 30 Enterococcus species isolates from stool samples were tested for the elaboration of virulence factors like hemolysin, gelatinase, biofilm production and the presence of the "esp" gene.
Virulence factors in Enterococcus isolates from stool samples
Comparison between virulence factors expressed by Enterococcus isolates from clinical samples and those from stool samples
Only two out of the thirty Enterococcus isolates from stool samples were found to be beta-hemolytic on human blood agar, whereas 33 out of the 157 clinical isolates were beta-hemolytic. However, the difference in the expression of this virulence factor by isolates from clinical samples and those present as commensals in stool was not found to be statistically significant [Table 4].
P value was found to be 0.0754 using Fisher's exact test.
Out of the 30 stool isolates, only one was found to be positive for gelatinase production, whereas 30 out of the 157 clinical isolates were found to be positive for gelatinase production. This difference between the clinical isolates and the isolates from stool was found to be statistically significant with a P value of 0.0324 (Fisher's exact test).
Ten out of the 30 isolates of Enterococcus from stool specimens were biofilm producers as tested by the microtiter plate method. There was no significant difference in the proportion of stool isolates producing biofilm and the proportion of clinical isolates producing biofilm (P = 0.0718 by Fisher's exact test).
"esp" gene was detected in only seven out of the 30 stool isolates, whereas a majority of the clinical isolates (45 out of 58) were positive for it. This difference was found to be statistically significant with a P value < 0.0001 using Fisher's exact test.
Enterococcus isolates from clinical specimens were, therefore, more likely to have the "esp" gene and produce gelatinase than isolates from stool samples.
| Discussion|| |
Although the propensity to acquire and spread genes responsible for antimicrobial resistance is the main reason for enterococci emerging as important nosocomial pathogens, the role of various other virulence factors in disease causation by these organisms cannot be overruled.
In our study, the expression of three virulence factors was evaluated -hemolysin, gelatinase and biofilm formation. In addition, an attempt was made to detect the presence of the "esp" gene in some isolates of Enterococcus.
Adherence to body surfaces is considered a major factor responsible for the pathogenicity of the clinical isolates of Enterococcus. Strains causing infection are considered to have a greater capacity to adhere to surfaces than commensal strains or strains isolated from food products. The "esp" plays a major role in the capability of enterococcal strains to form biofilms. "esp" was first described in a virulent gentamicin-resistant Enterococcus faecalis isolate.  Biofilm formation plays a major role in nosocomial infections like catheter-associated UTIs and even blood stream infections due to pacemakers. It also plays a vital role in endodontic infections. According to a study by Toledo-Arana et al. the biofilm-forming capability of enterococcal strains is confined to strains possessing the "esp" gene. Another study also gives out similar results after comparing the biofilm-forming capability of "esp"- positive and "esp"- negative strains. Natively "esp"-negative strains have been manipulated to express "esp" on their surface and were found to form biofilms after expressing "esp".  The "esp" gene and the "esp" encoded by it has been shown to enhance the persistence of Enterococcus species in urinary bladder and hence plays an important role in pathogenesis of UTIs.  In our study, 78.6% of the Enterococcus isolates from urine samples tested for "esp" gene were found to be positive for it (data not shown). However, all Enterococcus isolates were not tested for the presence of the "esp" gene and that is one of the limitations of this study. No significant difference was observed in the prevalence of the "esp" gene among vancomycin-sensitive strains of enterococci and vancomycin-resistant strains.
There was a significant difference in the proportion of "esp"- positive isolates from clinical specimens and "esp"-positive isolates from stool samples. Isolates of Enterococcus species from clinical specimens were more likely to possess the "esp" gene than isolates from stool samples. Such findings have also been reported by other workers. Shanker et al. showed a significant association of the "esp" protein with E. faecalis isolated from patients with UTI compared to fecal isolates.
Enterococci have been associated with biofilms on various kinds of indwelling devices like prosthetic heart valves, urinary catheters, artificial hip prostheses etc., and this capability to produce biofilms has been considered an important virulence factor of these organisms. Various methods like microscopic biofilm formation assay and epifluorescence microscopy have been tried to study the biofilm-forming capability of bacteria. However, the method that has been used very frequently in recent times is the microtiter plate biofilm production assay. This method is preferred because of its simplicity and cost-effectiveness. The microtiter plate biofilm assay technique was first devised for studying the biofilm-forming capability of Listeria monocytogenes by Djordjevic et al.  This method was later modified and used for studying biofilm formation in other gram-positive bacteria like coagulase-negative staphylococci and Enterococcus species.
In our study, apart from the microtiter plate assay, two other methods were evaluated for studying the biofilm formation by Enterococcus isolates - tube method and the Congo red agar method. Unlike the microtiter plate method, which is a quantitative biofilm assay, the tube method and the Congo red agar method are qualitative assays and their interpretations are often very subjective. The Congo red agar method has been evaluated before for studying biofilm formation in CONS isolates.  In our study, the sensitivity of the Congo red agar compared to the microtiter plate assay was found to be very low at 30%. The specificity of the assay was 77%. The sensitivity and specificity of the tube method was also low at 61% and 68%, respectively.
Fifty-three percent of the clinical isolates tested for biofilm production were found to produce biofilms in our study. Many studies have implicated the "esp" and the presence of the "esp" gene in biofilm formation by enterococci. , However, many studies have also reported results that indicate the contrary. Results obtained by Kristich et al. have demonstrated that in vitro biofilm formation is independent of the presence of the pathogenicity island harboring the "esp" coding sequence and depends mostly on environmental conditions. The results of our study seem to be in agreement with the latter view. The difference in the formation of biofilms by "esp"-positive and "esp"- negative strains was not found to be significantly different in our study. However, one limitation of our study was that the number of strains of enterococci tested for the presence of the "esp" gene was very less (58 strains).
Hemolysin production and gelatinase production was seen in 21% and 19% of all clinical isolates tested, respectively. Gelatinase elaborated by some Enterococcus isolates has been identified to be an extracellular zinc-endopeptidase capable of hydrolyzing gelatin, collagen, casein, hemoglobin and other peptides.  This virulence factor is mostly seen in isolates of E. faecalis. The role of gelatinase in causing endocarditis has been studied using animal models.  The role of gelatinase in enterococcal infection is in providing nutrition to the bacteria by degradation of host tissues.  Cytolysins (hemolysins) have been shown to be associated with increased mortality in animal (rabbit) models of enterococcal endocarditis.  Generally, the capability to produce the cytolysin (hemolysin) is plasmid mediated although occasionally, cytolysin genes can occur as chromosomal elements.  Cytolytic enterococci have been shown to be associated with bacteriocin production with activity against some gram-positive but not gram-negative bacteria.  Some bacteriocins produced by Enterococcus (known as Enterocins) are active against Listeria species, Clostridium species and even Staphylococcus aureus. 
We compared the expression of virulence factors in vancomycin-resistant and vancomycin-sensitive strains of enterococcus species. No statistically significant difference was seen between the two groups for any of the virulence factors. Few other studies have reported similar findings. 
Enterococcus species isolates from stool samples were compared with the clinical isolates for the elaboration of virulence factors. While there was no statistically significant difference between clinical isolates and stool isolates with regards to hemolysin production and biofilm production, gelatinase production was found to be significantly more among clinical isolates. Clinical isolates were also more likely to possess the "esp" gene when compared to the isolates from stool samples.
Although many putative virulence factors of enterococci have been described, their association with an adverse patient outcome or patient mortality is still not accepted by all workers in the field and there have been studies that point to the contrary. A study done on enterococcal bacteremia studied the relationship of virulence factors like hemolysin production, gelatinase production and presence of the "esp" with patient mortality. It was concluded that the virulence factors studied were not associated with increased 14-day mortality rate among patients with bacteremia.  In our study, we have not evaluated the association between the elaboration of virulence factors by Enterococcus strains and patient outcome.
| Conclusion|| |
Although a majority of the clinical isolates of enterococci tested were found to be positive for biofilm production in our study, contrary to some other studies, this biofilm forming ability was found to be independent of the presence of the "esp" gene. The tube method of biofilm detection and Congo red agar had low sensitivity and specificity when compared to the microtiter plate method of biofilm detection. No significant association between the expression of virulence factors and vancomycin resistance was found in the study. Among the virulence factors, gelatinase production and the presence of the "esp" gene was seen more commonly in clinical isolates than commensal strains of Enterococcus, whereas there was no significant difference between the commensal strains and clinical isolates with regards to the elaboration of hemolysin and biofilm formation.
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Department of Microbiology, JIPMER, Puducherry
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
[Table 1], [Table 2], [Table 3], [Table 4]
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