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Year : 2020  |  Volume : 38  |  Issue : 2  |  Page : 200--209

Biofilm synthesis and other virulence factors in multidrug-resistant uropathogenic enterococci isolated in Northern India

Ayan Kumar Das1, Mridu Dudeja1, Sunil Kohli2, Pratima Ray3, Manvi Singh4, Preet Simran Kaur1,  
1 Department of Microbiology, Hamdard Institute of Medical Sciences and Research, Jamia Hamdard, New Delhi, India
2 Department of Medicine, Hamdard Institute of Medical Sciences and Research, Jamia Hamdard, New Delhi, India
3 Department of Biotechnology, School of Chemical and Life Sciences, Jamia Hamdard, New Delhi, India
4 Department of Pharmaceutics, School of Pharmaceutical Education and Research, Jamia Hamdard, New Delhi, India

Correspondence Address:
Mr. Ayan Kumar Das
Department of Microbiology, Hamdard Institute of Medical Sciences and Research, Jamia Hamdard, New Delhi - 110 062


Purpose: Enterococci express high degree of resistance towards wide range of antibiotics. Production of biofilm and many virulence factors along with drug resistance makes it difficult to eradicate the infection from urinary tract. The present study detected the expression of such factors including biofilm production by multidrug-resistant (MDR) enterococci. Materials and Methods: Drug susceptibility of 103 uropathogenic enterococci was performed followed by estimation of minimum inhibitory concentration of high-level gentamicin and vancomycin by microbroth dilution method. Vancomycin-resistant genes were detected by multiplex polymerase chain reaction. Production of virulence factors such as haemagglutination, caseinase, lipase, gelatinase, haemolysin and β-lactamase was detected by phenotypic methods in MDR strains. Biofilm production was detected by calcofluor-white fluorescence staining and semi-quantitative adherence assay. Results: 45% and 18.4% of the isolates were high-level gentamicin-resistant and vancomycin-resistant enterococci (VRE), respectively. vanA gene was detected in 14 and vanB gene in 5 strains. Biofilm, caseinase and gelatinase were the most expressed virulence factor. Expression of caseinase, gelatinase and lipase was significantly higher in Enterococcus faecalis (P < 0.05). Expression of haemagglutination, gelatinase and haemolysin among the vancomycin-resistant isolates was significantly higher (P < 0.05). Conclusion: VanA and vanB are the prevalent genotypes responsible for vancomycin resistance. The high prevalence of MDR enterococcal strains producing biofilm and virulence determinants raises concern. asa1, hyl, esp, gelE, cyl and other genes are known to express these factors and contribute to biofilm formation. Most uropathogenic enterococci expressed biofilm at moderate level and can be detected effectively by calcofluor-white staining. No correlation was noted between vancomycin resistance and biofilm production.

How to cite this article:
Das AK, Dudeja M, Kohli S, Ray P, Singh M, Kaur PS. Biofilm synthesis and other virulence factors in multidrug-resistant uropathogenic enterococci isolated in Northern India.Indian J Med Microbiol 2020;38:200-209

How to cite this URL:
Das AK, Dudeja M, Kohli S, Ray P, Singh M, Kaur PS. Biofilm synthesis and other virulence factors in multidrug-resistant uropathogenic enterococci isolated in Northern India. Indian J Med Microbiol [serial online] 2020 [cited 2020 Dec 1 ];38:200-209
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Enterococci are responsible for a large percentage of Gram-positive urinary tract infections (UTI) Worldwide.[1] The pathogenicity of Enterococcus is attributed to the presence of wide range of virulence factors, and it is intrinsic as well as acquired transferable drug resistance characteristics.[2] Acquired resistance towards high-level gentamicin and vancomycin is a serious concern as the therapeutic options becomes extremely limited. In India, 5% of all multidrug-resistant (MDR) pathogen-associated deaths have been ascribed to enterococci.[3]

Biofilm production is one of the most important virulence factors and plays a crucial role in pathogenesis of enterococci.[4] 25% of all catheter-associated UTI have been linked to biofilm producing enterococci.[5] Biofilms are mono- or poly-microbial aggregations irreversibly attached to a substratum or interface or to each other and surrounded in a matrix of self-produced extracellular polysaccharide along with abiotic components from the environment. Within the aggregation, organisms communicate with each other through cell-to-cell signalling mechanisms such as quorum sensing.[6] The presence of persister cells in the matrix and decreased penetration of antibiotics through biofilm causes antibiotic tolerance and may lead to chronic infections.[5] As the biofilm gets older, the tolerance rises, in turn increasing the minimum biofilm eradication concentration.[5] UTI by biofilm producing bacteria is difficult to eradicate than infection caused by a non-biofilm producer.[7]

Pathogenic species of enterococci express many other virulence factors along with biofilm. Expression of many factors such as adhesins, gelatinase (gelE), Enterococcus surface protein (Esp), aggregation substances and cytolysins (cylA) along biofilm formation enhances the virulence of enterococci by many folds.[8] These factors enhance the ability of the pathogen to invade, attach and survive through the acquisition of nutrients in the host tissue.[8] Their presence in drug-resistant strains increases the severity of the infection.[9],[10]

Information related to the prevalence and presence of virulence factors including biofilm production may help in selecting the best treatment strategies among the already limited options and may help in understanding the complex pathogenesis of the organism. Even though the rate of UTI by MDR bacteria has increased over the years, only limited data from India are available regarding expression of virulence factors. The aim of the present study was to determine the prevalence of vancomycin resistance and to evaluate the phenotypic expression of biofilm and virulence factors such as gelatinase, caseinase, lipase, haemolysin and haemagglutination in MDR enterococci causing UTI.

 Materials and Methods

This cross-sectional study was conducted between January 2016 and May 2018 at microbiology laboratory of a tertiary care hospital in Northern India. A total of 103 enterococcal strains with colony-forming unit (CFU) count over 105/ml, isolated from culture of aseptically collected midstream urine samples from symptomatic cases of UTI, were considered for the study and further processed. The isolates were identified on the basis of standard cultural and biochemical properties.[11]

Antimicrobial susceptibility testing for the enterococcal isolates was performed by disc diffusion method as recommended by Clinical Laboratory Standards Institute (CLSI).[12] A reference strain of Enterococcus faecalis ATCC 29212 was used as a control. The enterococcal species were identified using GP-ID card in VITEK-2 system (Biomerieux, France). MIC of high-level gentamicin and vancomycin was determined by microbroth dilution method as per CLSI guidelines.[12] Isolates which were resistant to HLG and/or vancomycin along with at least one agent of two or more antimicrobial groups were further evaluated for production of virulence factors.

Isolates were streaked onto brain–heart infusion (BHI) agar (HiMedia, Mumbai) with 5% sheep blood. Beta-haemolysis around the individual colonies after 16–24 h incubation at 37°C was considered as haemolysin production.[13]

Haemagglutination was detected by slide agglutination method. 2–3 colonies of an isolate were mixed with a drop of 3% human and sheep RBC in phosphate-buffered saline on a clean glass slide. Agglutination of RBC within 5 min was considered as positive.[14]

To detect caseinase production, colonies were streaked onto Muller­Hinton agar (HiMedia, Mumbai) with 3% skimmed milk and the plate was incubated at 37°C for 16–24 h. Appearance of transparent zone is taken as positive for casein hydrolysis.[15]

β-lactamase production was detected by nitrocefin test as per CLSI guidelines.[12] A 0.5 mM nitrocefin solution was prepared using nitrocefin powder (Merck Life Sciences, Mumbai). 20 μl of bacterial suspension was mixed with the same amount of nitrocefin solution on a glass slide, and appearance of red colour within 1–2 min was considered as β-lactamase positive.[16]

Nutrient agar (HiMedia, Mumbai) supplemented with 3% gelatin in Bijou bottle was stab inoculated with the isolates and incubated at 37°C. After 24 h, the bottles are then refrigerated at 4°C for half an hour. Liquefaction was considered as positive gelatinase production.[17]

Lipase production was detected using egg yolk agar, prepared using 1% fresh hen's egg yolk in nutrient agar (HiMedia, Mumbai). The isolates were inoculated on medium and incubated at 37°C for 24 h. A wide zone of opalescence around the colonies was considered as positive.[14]

Biofilm assay

Calcofluor-white staining

The strains were mixed in BHI broth (HiMedia, Mumbai) with 2% glucose and incubated overnight at 37°C. The broth was diluted 1:100 and taken in a sterile 50 ml beaker, and a clean, sterile grease-free coverslip was placed inside. The beaker was incubated at 37°C for 24 h without agitation. The coverslip was gently taken out and was washed with phosphate-buffered saline to remove free planktonic cells.[18] It was then placed in hot air oven at 60°C for 1 h for heat fixing. The coverslip was then placed on a glass slide and stained with 0.02% calcofluor white (Sigma-Aldrich, USA). The slide was incubated in dark at room temperature for 20 min. It was then observed at ×40 magnification of fluorescence microscope (CX20i with FLUOLED Easy illuminator attachment [480 nm], using 535 nm barrier filter). Fluorescence emitted confirmed the production of exopolysaccharides.[19]

Semi-quantitative adherence assay-microtitre plate method

Each isolates were mixed in BHI broth with 2% glucose and incubated overnight at 37°C. The broth was diluted 1:100 and 200 μL was pipetted into sterile flat-bottomed 96-well microtitre plates (Tarsons, Kolkata) in triplicate. Un-inoculated BHI broth was put in eight wells per tray as negative control. Plate was then incubated at 37°C for 24 h. Post incubation, the broth was carefully taken out of the well with the help of micropipette. The wells were then washed thrice with 200 μl phosphate-buffered saline and kept at 60°C for 1 h. The biofilms were then stained with 200 μl of 1% aqueous crystal violet solution [2],[8],[20],[21] for 15–30 min and washed with phosphate-buffered saline to remove the excess stain. The plate was kept inverted for drying. 200 μl of ethanol-acetone (80:20 mixture) was added to each well for solubilising the bound crystal violet.[2],[21]

Absorbance was measured at 570 nm using a microtitre plate reader (MAGO-4, Transasia, India). Cut-off was calculated as mean OD negative control added to 3 times the standard deviation (Nc mean OD + 3X SD). OD values above the cut-off were considered positive for biofilm production. The positive isolates were categorised as weak biofilm producer (OD > cut-off, ≤2 × cut-off), moderate biofilm producer (OD > 2 × cut-off, ≤4 × cut-off) or strong biofilm producer (OD ≥ 4 × cut-off).[22]

The presence of vanA, vanB, vanC, vanD, vanE and vanG genes was detected by multiplex polymerase chain reaction (PCR) using previously described primers.[22],[23],[24] [Table 1] depicts the primer sequences. For DNA extraction, 3–5 colonies from overnight growth on sheep blood agar was taken and suspended in 1 ml of sterile-distilled water. The suspension was heated to 100°C for 15 min and was then centrifuged at 15,000 g for 10 min. The DNA-containing supernatant was used as a template. Multiplex PCR was performed in 2720 thermal cycler (Applied Biosystems) and was programmed for initial denaturation step at 94°C for 2 min, followed by 25 cycles of amplification (94°C for 60 s, 55°C for 60 s and 72°C for 60 s), and an extension at 72°C for 5 min. PCR products were resolved by electrophoresis on a 1.5% agarose gel stained with ethidium bromide on Sub-Cell GT electrophoresis system (Bio-Rad, USA). A 100-bp DNA ladder (Vivantis Technologies Sdn. Bhd., Malaysia) was run in each gel, and the vancomycin-resistant enterococci (VRE) genotype was determined by the size of the amplified product.{Table 1}

Data generated were analysed using the Statistical Package for the Social Sciences (SPSS), version 25, IBM SPSS Inc, USA. Significance was determined among variables using Chi-square test. Significance is defined as P ≤ 0.05.


A total of 103 enterococcal strains with count more than 105 CFUs per ml of urine were isolated and processed further. Among them, 78.6% were E. faecalis and 17.4% were Enterococcus faecium. Two Enterococcus avium and one strain each of Enterococcus gallinarum and Enterococcus durans was also isolated.

Antibiotic resistance

The antibiotic sensitivity results showed high resistance towards beta-lactam drugs. Most isolates were also resistant to erythromycin, tetracycline and fluoroquinolones. The least resistance was seen towards linezolid [Table 2]. Microbroth dilution confirmed that nearly 45% of isolates were high-level gentamicin resistant (HLGR) (MIC ≥500 μg/ml). The percentage of high-level gentamicin resistance was much higher among E. faecium than E. faecalis. This was found to be statistically significant (P < 0.05). Similarly, the incidence of amoxicillin-clavulanic acid resistance was significantly higher among E. faecium than E. faecalis (P < 0.05). Vancomycin resistance was seen among 18.4% of isolates. The difference in incidence among the two major species was not statistically significant. Phenotypically, 14 isolates appeared to have vanA gene as they were resistance to both vancomycin and teicoplanin. Five isolates appeared as vanB type, as they were sensitive to teicoplanin.{Table 2}

Expression of virulence factors

Fifty isolates, which were resistance to at least 3 different groups of antibiotics, were further tested for virulence factors. [Table 3] depicts all the strains, divided as per number of antibiotics they are resistant to and the virulence factors produced. None of the MDR strains showed beta-lactamase production. Five strains showed resistance to six groups of antibiotics and produced at least 5 types of virulence factors. Two strains produced no virulence factor and were resistant to 3 groups of antibiotics.{Table 3}

Among all MDR isolates, biofilm, caseinase and gelatinase were the most expressed virulence factor (82%, 72% and 66%, respectively). Haemagglutination with human RBC was seen in 14% of the cases. Only 1 MDR isolate agglutinated sheep RBC. 40% and 38% of isolates were positive for haemolysin and lipase production, respectively [Figure 1].{Figure 1}

There was no difference in biofilm production by MDR strains of E. faecalis and E. faecium (P > 0.05). However, in case of other factors, MDR E. faecalis had a much higher percentage of expression and the difference was significant in case lipase, caseinase and gelatinase (P < 0.05). Out of the 50 MDR strains tested for virulence factors, 19 were vancomycin resistant. [Figure 2] compares the expression of virulence factors by these VRE with MDR vancomycin-sensitive enterococci (VSE). There was no significant difference in lipase, caseinase and biofilm production between the two groups. Expression of haemagglutination, gelatinase and haemolysin by the vancomycin-resistant group was significantly higher (P < 0.05).{Figure 2}

Biofilm production was detected by calcofluor-white staining method [Figure 3]. The emission of greenish fluorescence when observed under UV microscope confirmed the production of exopolysaccharides by the isolates. The results were similar as obtained by the semi-quantitative adherence assay. Out of the 50 isolates tested, 41 produced biofilms in vitro. Most isolates showed moderate level of biofilm production [Figure 4]. It was observed that most non-biofilm-producing strains showed lesser positivity towards other virulence factors as well [Table 3].{Figure 3}{Figure 4}

Detection of vancomycin resistance genes

VanA and VanB were the predominant genotypes detected by PCR. Other vancomycin resistance genes were not detected. Fourteen strains carried VanA gene whereas VanB gene was found in five isolates. Among the VanA strains, 11 were E. faecalis and 3 were E. faecium. VanB was present in 3 E. faecalis and 2 E. faecium strains [Table 4].{Table 4}


Enterococcus has emerged as an important Gram-positive urinary pathogen in last few decades. Even though many species of enterococci have been associated with UTI, E. faecalis has been found to be responsible for nearly 90% of the cases, whereas E. faecium remains distant second.[25] The finding was similar is the present study. Most isolates were resistant to beta-lactam drugs. This was in accordance with other studies conducted in this region and elsewhere.[22],[26] Similar to this report, high resistance towards fluoroquinolones, erythromycin and tetracycline was observed in studies done by Goel et al. in Delhi [27] and by Mathur in Rajasthan.[28]

The isolation rate of HLGR enterococci was similar to recent studies conducted in neighbouring areas.[22],[28] Comparatively, much higher percentage of HLGR among uropathogenic enterococci was reported from southern state of Karnataka in the same year.[29] Vancomycin resistance was seen in 18.4% of isolates. It was slightly higher than the other study undertaken in 2014 at Delhi.[27] VRE express low affinity cell wall receptors mediated due to presence of one or more resistant genes. Nine glycopeptide-resistant genes vanA, vanB, vanC, vanD, vanE, vanG, vanL, vanM and vanN are known, the last three been identified more recently.[30] Most Indian studies have reported vanA genotype as the most prevalent one. In 2016, Tripathi et al. found vanA gene in all 118 strains.[31] Another study in 2017 detected vanA gene in 57 isolates and vanC1 in 6 E. gallinarum.[32] vanB in 16 isolates and vanA in 20 isolates were reported by Biswas et al.[33] A study conducted in Beijing, China, found van A gene in all the E. faecium strains tested. Among the 3 E. faecalis strains, one carried vanB gene.[34] Between 2005 and 2019, Hammerum et al. evaluated 2664 VRE strains in Denmark, out of which 93.9% were vanA gene carrying E. faecium.[35] vanA and vanB gene confer acquired inducible high-level resistance. The present study substantiates these two as the more prevalent vancomycin-resistant genotype in the study region.

Taken as a whole, E. faecium showed higher resistance towards various antibiotics than E. faecalis. Nearly 50% of the isolates were MDR. Bhatt et al. reported 63% MDR enterococci in 2015.[36] Shrestha et al. reported 66.67% of enterococci as MDR in urine from the neighbouring country Nepal.[37] In concurrence to most other studies from India, linezolid was found to be the most effective drug.[27],[28],[31] Less resistance was also showed towards nitrofurantoin and these two drugs can be the affectively used for treatment of UTI by MDR enterococci in this region.

None of the enterococcal isolates showed β-lactamase production. β-lactamase is rarely expressed by enterococci.[12] However, enterococci show resistance towards β-lactam drugs by virtue of other mechanisms such as increased expression of low-affinity penicillin-binding proteins.[38] This may be the reason for high level of resistance towards penicillin and amoxicillin in the present study even without expression of β-lactamase. The first β-lactamase-producing E. faecalis was reported from the USA in 1981 and very infrequently thereafter.[38] Agarwal et al. reported one such case from India in 2009.[39]

Haemagglutination properties are indicative of adherence capacity of the strains to receptors in the urinary tract. Enterococci express many different adhesin molecules such as aggregation substance (AS), Enterococcus surface protein (Esp), adhesion of collagen (Ace), E. faecalis antigen A (EfaA) and endocarditis and biofilm-associated pili (Ebp) on its surface [40] that help in binding with the host epithelium.[8] The variation in agglutination properties to RBC of difference species may arise due to variation in adhesion molecules among different strains. In the present study, just one isolate agglutinated sheep RBC. Similar to this, Gulhan et al. from Turkey reported no agglutination with sheep RBC.[41] Another Indian study reported equal haemagglutination with sheep and human RBC.[14] The study conducted by Furumura et al. from Brazil reported haemagglutination of rabbit RBC by 40.6% of the isolates.[42] As high as 87% of E. faecalis were found to be haemagglutination positive in the study done by Kadhum et al. in Iraq.[43]

Strains producing gelatinase are better equipped to extract nutrients by degrading host tissue. It is a secreted protease coded by the gelE gene.[40] GelE is a plasmid-coded extracellular metalloprotease that cleaves gelatin, collagen and fibrin, haemoglobin, C3 component of complement and casein.[20],[44] Studies have shown that a strain may carry gelE gene but can be phenotypically negative for the enzyme. Increased expression of gelatinase has been reported in hospital-acquired strains.[45] Two-thirds of all isolated tested in this study expressed gelatinase. Contrary to this, the study conducted in Varanasi reported only 6.08% gelatinase production.[2] None of the isolates included in the study by Furumura et al. expressed gelatinase.[42] On the other hand, 60% of the strains expressed gelatinase in the study by Comerlato et al.[8] As per the polish study done by Strzelecki et al., 71% of the uropathogenic enterococci were gelatinase producers.[46] Patidar et al. reported 34.6% gelatinase production.[15]

Another factor that was expressed by most MDR strains included in this study was caseinase. Not many India studies have undertaken phenotypic detection of caseinase enzyme. Corroborating with the result of the present study, Patidar et al. reported 73% of the strains as caseinase producers.[15] Biswas et al. found only 30.6% of the strains as caseinase positive.[14] Among the studies done in other countries, Dada et al. found 15% strains positive for caseinase in Malaysia.[47] The study from Brasil by Furumura et al. reported Caseinase activity by 75% of the isolates.[42]

Expression of lipase was seen in only one-third of the strains in the present study. Biswas et al. recorded lipase production by 11.1% of the strains tested.[14] In the 2014 Polish study by Dworniczek et al., 24% of the E. faecalis strains isolated from cases of UTI were found positive for lipase production. Among E. faecium, the rate was 8%.[48] Furumura et al. found 71.9% of E. faecalis positive for lipase.[42] One more Brazilian study of 2016 done by Komiyama et al. tested enterococci isolated from oral cavity for virulence factors and found 92% of them to be lipase producers.[49]

Haemolysin is a cytolytic toxin and is bactericidal against other Gram-positive bacteria. Cytolysins can lead to lysis of human RBC, WBC and intestinal epithelial cells.[44] The production of cytolysin is regulated by the cylLs group of genes located on pheromone-responsive plasmids. Clinical isolates carry these genes in higher frequency.[42] The observed rate of haemolysin production by isolates examined in this present study corroborates to finding of other reports.[2],[13] Studies conducted by patidar et al.[15] and Manavalan et al.[50] reported relatively higher and lower rate of haemolysin production, respectively. Gulhan et al. reported cytolysin in 15.3% of the isolates only.[41] Aladarose et al. found only 12.9% of the isolates β-haemolytic, using 5% horse blood agar.[44]

The present study compared expression of virulence traits between VRE and VSE strains and found significantly higher expression of haemagglutination, gelatinase and haemolysin among VRE. No difference in expression of factors between the two groups was identified in the study by Comerlato et al.[8] The study by Praharaj et al. too could not find any significant difference in expression of gelatinase, haemolysin and biofilm.[13] Aladarose et al. reported 91.6% of vancomycin-resistant (VRE) strains as strong/moderate biofilm producer.[44] Contrary to this, Shridhar et al. found all the VRE isolates tested to be biofilm non producers.[51] Banerjee et al. reported significantly higher expression of all virulence factors among VSE isolates.[2]

Most MDR strains tested were biofilm producers in the present study. Among the isolates tested by Banerjee et al., only 26.12% produced biofilm.[2] Manavalan et al. reported 73.81% biofilm producers.[50] Many other studies reported similarly high rate.[14],[15] It was observed in the present study that the biofilm non-producing strains expressed lesser number of other virulence factors [Table 3]. Moreover, such strains were resistant to lesser number of antibiotic groups in comparison to biofilm producers. Studies have shown that virulence factors, biofilm production and antibiotic resistance are interrelated. Enterococcal surface adhesin like aggregation substance plays an important role in biofilm formation. Expression of Esp regulates primary adherence and biofilm formation in enterococci.[52] Adherence of the organism to the bladder wall is facilitated by Esp through mucin and uroplakin receptors.[2] Esp helps in evading the immune system and colonisation and establishment of UTI.[8],[44] High-molecular-weight eDNA, which is a vital constituent of biofilms, is released by GelE-regulated autolysis. GelE is also known to contribute directly in bacterial adherence and biofilm formation.[20] Few studies have shown that cytolysins may be associated with biofilm formation in case of UTI.[20],[44] Biofilm confers antibiotic tolerance due to low penetration.[5] The drug-resistant genes are regulated differentially in a biofilm-embedded enterococcus than a planktonic cell. Due to the phenomenon of intra- and inter-species horizontal gene transfer, enterococcal biofilms acts as a gene reservoir for transmission of antimicrobial resistance.[5]

The present study does not indicate any relationship between the species and biofilm formation as was observed by Banerjee et al., where no significant difference in biofilm production was observed between E. faecalis and E. faecium.[2] However, other studies from India, the USA, Britain, Italy and Japan had reported higher expression of biofilm by E. faecalis over E. faecium,[14],[20] and an extended study with larger sample size is warranted to establish this association among isolates of this region.

Two methods were used in this study for biofilm detection and they yielded same result. The biofilm on coverslip was stained with calcofluor white which binds to glycosidic bonds β-(1-3) and β-(1-4) of exopolysaccharides in biofilm.[19] The method is quite simpler and less time-consuming. Only a handful of studies have used this method for visualisation of enterococci biofilm and should be employed more.

In accordance with many similar studies, the microtitre plates were incubated for 24 h before analysing for biofilm formation.[2],[21] Most isolates produced moderate level of biofilm, similar to the findings reported by Comerlato et al. and others.[8],[14],[53] However, it has been reported that some strains take up to 48 h for irreversible attachment.[54] A longer incubation may have altered the result in terms of biofilm strength. Conforming to the present work, BHI broth supplemented with 0.25%–2% glucose has been used in many studies as a medium for biofilm growth.[2],[15],[21] Few others have used Tryptic soy broth (TSB)[4],[13],[17],[20] and lysogeny broth (LB).[18] It has been reported that BHI broth yields more consistent result for Enterococcus biofilm formation in comparison to TSB which is better for staphylococcal studies.[21],[53]

The high positivity of biofilm production among MDR strains underlines the need for better drug selection. For most antibiotics, the MIC and minimum bactericidal concentration (MBC) for biofilm bacterial cell are 10–1000 times higher than that required for planktonic cells. Such high concentration is impossible to achieve in vivo due to due to toxicity.[55] Hence, drugs which are biofilm active and have better penetration should be selected while treating enterococcal infections in this region. Usually, erythromycin, clindamycin, tetracycline, fluoroquinolones and linezolid penetrate better than beta-lactam, aminoglycosides and glycopeptide antibiotics.[55]

Studies have proven microtitre plate method as the most common and effective procedure for in vitro detection of biofilm.[53] The present study detected phenotypic expression of biofilm and not the genetic markers as done in few other studies. This can be considered as a limitation. A number biofilm-forming genes, for example, asa1, pilA, pilB, hyl, esp, fsr, efaAfm, gelE and cyl, were described and used previously.[20] Scholars have assessed the correlation between presence of these genes and biofilm formation capacity in Enterococcus strains. While some studies have showed association of these genes with biofilm formation, many others have failed to establish any positive relationship.[7],[20],[44],[53] The study will be widened further involving these genetic markers to evaluate any relationship with biofilm formation.

Overall, the study endorses E. faecalis over E. faecium as the species with higher expression rate for most of the virulence factors. Many other studies have shown that E. faecalis may harbour many virulent gene that may be absent in E. faecium.[40] Comerlato et al. have reported similar findings.[8] This high virulence expression may be one of the reasons for higher rate of isolation of E. faecalis from clinical specimens. In this study, each MDR strains expressed at least 3 virulence factors on average. These findings emphasise on the need of diligent and regular surveillance, as most the drug-resistant traits and virulence genes in enterococci are plasmid borne with high capacity of intra- and inter-genus genetic exchange. Few studies have reported no relationship between vancomycin resistance and virulence factors.[8],[56] However, many articles have postulated that there may be decrease in virulence with increase in drug resistance. A study by Banerjee et al. showed decreased expression of virulence factors in vancomycin-resistant strains.[2] The present study, however, disproves the irrefutability of such assumption as the VRE strains showed higher expression of virulence factors.


The presence of virulence factors in opportunistic pathogens such as enterococci helps them in establishing serious infections at susceptible site like the urinary tract. Genes such as asa1, hyl, esp, fsr, efaAfm, gelE, cyl and few more are known to express factors such as adhesins, caseinase, gelatinase, lipase and haemolysin and also collectively influence biofilm formation. Since most MDR strains were found to be also biofilm producers in this study, the effective dosage of prescribed drug may vary. Vancomycin remains an important option for treatment of MDR isolates but incidence by VRE is increasing. VanA and VanB are the prevalent genotype in this region. β-lactams, fluoroquinolones and macrolides may not be effective on isolates from this territory. Nitrofurantoin can be effectively used for treatment of enterococcal UTI, and linezolid is a valuable choice for MDR strains including VRE. Calcofluor-white staining method is a cheap, easy and effective method for detection of biofilm and should be employed more often. With increase in importance of Enterococcus as a potent pathogen, the recognition of factors associated with invasiveness and disease progression has become important and a topic that warrants further evaluation.

Financial support and sponsorship

This was a self-funded study.

Conflicts of interest

There are no conflicts of interest.


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