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Improved detection of esp, hyl, asa1, gelE, cylA virulence genes among clinical isolates of Enterococci

BMC Research Notes volume 13, Article number: 170 (2020) Cite this article

Abstract

Objective

Virulence factors (VFs) among the clinical strains of enterococci play a vital role in pathogenesis. This study was aimed to screen for cylA, asa1, gelE, esp and hyl among Enterococcus faecalis (n = 89) and E. faecium (n = 51) by multiplex PCR. The previously reported multiplex PCR was modified to 2 duplex (asa1 and gelE, cylA and esp) PCRs and 1 simplex (hyl) PCR. The idea of the modification of the multiplex PCR proposed here emerged in the course of the research study when majority of the isolates which phenotypically exhibited virulence traits were found to be negative for the respective gene.

Results

cylA, gelE and asa1 were significantly predominant in E. faecalis (59.55%, 85.39%, 86.51%) than E. faecium (1.96%, 60.78%, 9.80%) (p < 0.0001, p = 0.001967, p < 0.0001). hyl was detected in E. faecium (5.9%) only. The number of VFs detected in each isolate was recorded as the VF score. E. faecalis isolates had a VF score pattern of score 4 (34.83%), score 3 (26.96%), score 2 (28.08%) and score 1 (8.98%) while E. faecium had score 4 (1.96%), score 3 (7.84%), score 2 (25.49%) and score 1 (41.18%). This modification of the PCR protocol could resolve the problem of decreased detection of virulence determinants in enterococci.

Introduction

Enterococcus faecalis and E. faecium, the two most common species of enterococci that inhabit the gastrointestinal tract are a leading cause of opportunistic and nosocomial infections in humans. Pathogenesis of enterococci is attributed to an array of virulence factors (VFs) viz., aggregation substance (AS), gelatinase (Gel), cytolysin (Cyl), enterococcal surface protein (Esp) and hyaluronidase (Hyl).

Cytolysin elaborated by hemolytic strains of E. faecalis contributes to virulence in animal models and in human infections [1,2,3]. The cytolysin operon is a two-component system, lysin (L) encoded by cylL1, cylL2, cylM, cylB and an activator (A) encoded by cylA [4, 5]. Gelatinase encoded by gelE, is an extracellular zinc-endopeptidase/protease produced by E. faecalis that is capable of hydrolyzing gelatin, collagen, casein, hemoglobin, and other peptides [6]. Gelatinase production in E. faecalis contributes to virulence in animals and humans [7, 8]. Gelatinase damages the host tissue facilitating bacterial migration and spread [9], colonisation and persistence by biofilm formation [10]. Enterococcal surface protein, encoded by esp, is significantly higher among clinical isolates than faecal isolates and is associated with increased virulence [11], colonization and persistence in the urinary tract [12] and biofilm formation [13]. Aggregation substance, encoded by asa1, facilitates the conjugative transfer of sex pheromone gene-containing plasmids [14] and enhances virulence (adherence to renal tubular cells [15], heart endocardial cells [16] and internalization by intestinal epithelial cells [17]). Hyaluronidase, encoded by the chromosomal hyl, is reported to be specific for E. faecium [18, 19] and shows homology to the hyaluronidases of other Gram positive cocci [20]. Esp and Hyl are known to be specific for E. faecium, while AS, Gel, Cyl, Esp for E. faecalis [18, 19].

Though phenotypic and genotypic methods are available for the detection of VFs, majority of the previous studies (Additional file1: Table S1) have adopted the multiplex PCR protocol described by Vankerckhoven et al. [18]. Nevertheless, in our experience, we found isolates which phenotypically exhibited virulence traits were found to be negative for the respective gene. In addition, non-specific amplifications were observed. Hence, this study was designed with slight modifications to the existing multiplex PCR protocol [18].

Main text

Materials and methods

Clinical samples were collected after obtaining approval from the Institutional Ethical Committee, Sree Balaji Dental College & Hospital, BIHER, Chennai, India (IEC No: SBDCECM106/14/08/dt19.06.2014). A total of 140 clinical isolates of Enterococci (E. faecalis [n = 89], E. faecium [n = 51] from urine [n = 111], pus [n = 24], body fluids [n = 4] and blood [n = 1]) from patients with urinary tract infection, pyogenic wound infection, infected body fluids, bacteraemia attending tertiary care hospitals in Chennai, South India were included in the study. Species identification and characterization was performed as per standard biochemical tests [21] and further confirmed using Enterococcus Differential Agar supplemented with 1% 2,3,5-Triphenyl Tetrazolium Chloride (TTC) (HiMedia Laboratories Pvt Ltd, Mumbai, India).

Phenotypic screening of virulence

Hemolysin production was assessed using blood agar plates (5% defibrinated sheep blood). A clear zone of haemolysis around enterococcal colonies after incubation at 37 °C for 24 h was scored positive [22]. Gelatinase production was detected by stabbing enterococcal isolates into 12% gelatin and after incubation at 37 °C for 24 h, positive gelatinase activity was indicated by liquefied gelatin even after refrigeration at 4 °C for 4 h [22]. Slime production was detected using Congo Red agar. A positive slime layer formation was indicated by black pigmented enterococcal colonies after incubation at 37 °C for 24 h [23].

Multiplex PCR

DNA was extracted from overnight pure cultures of Enterococcal isolates by boiling lysis method. All Enterococci strains were screened for the presence of five VFs encoding genes (asa1, cylA, esp, gelE and hyl) using multiplex PCR as previously described [18]. Five primer pairs were used to amplify the genes asa1, gelE [18], cylA [24], esp [25] and hyl [18]. All the primers used in the study were synthesised at Macrogen (South Korea). This multiplex PCR is the most commonly used protocol for screening of virulence genes among enterococci (Additional file 1: Table S1). However, in our study, a very low prevalence of the virulence determinants was detected. Isolates which phenotypically exhibited the virulence trait was found to be negative (gene not detected by the multiplex PCR protocol) for the respective gene. In addition, non-specific amplifications were observed, amplicon size were not specific to the one indicated in the reference article [18]. Hence, simplex PCR for the individual genes were performed, followed by PCR with all possible combinations of the 5 genes. Finally, the following combinations of PCR reactions were standardised.

PCR standardization

Three different PCR reactions were standardised [two duplex (asa1 and gelE; cylA and esp) and one simplex (hyl) PCR]. Each 25 µl PCR reaction was set up with 2 µl of DNA template, 10× PCR buffer containing 15 mM MgCl2, 10 pmol of each primer specific for the respective gene (for duplex 1: asa1, gelE, duplex 2: cylA, esp and simplex: hyl (Macrogen, Korea), 0.5 U (for duplex 1: asa1, gelE and simplex: hyl) and 1U (for duplex 2: cylA, esp) of TaqDNA polymerase (Genet Bio Co, South Korea), 10 mM of each dNTP (BioBasic, Canada Inc) and 100 mM MgSO4 (New England BioLabs Inc, USA).

The cycling conditions include an initial denaturation at 95 °C for 5 min, followed by 30 cycles of denaturation (94 °C for 1 min), annealing (56 °C for 1 min), and extension (72 °C for 1 min), and a final extension for 8 min at 72 °C. PCR was carried out in Veriti™ 96-well Thermal Cycler, Applied Biosystem, USA. Known positive and negative controls were included for each run. DNA ladder, 100-bp (GeNet Bio, South Korea) was included as a molecular size marker.

DNA sequencing of virulence genes

PCR amplicons of each gene from representative isolates were purified by FavorPrep GEL/PCR Purification kit (Favorgen, Taiwan) and sequenced by Sanger sequencing method at Macrogen (South Korea) in single directions by respective forward primer using ABI PRISM®BigDye™ Terminator and ABI 3730XL sequencer (Applied Biosystems, USA). All the virulence gene sequences were compared with known sequences in NCBI Database by using BLAST analysis (http://www.ncbi.nlm.nih.gov/BLAST/) and the sequences were deposited in the NCBI GenBank database. (GenBank Accession numbers: MN398378 (asa1), MN398379 (cylA), MN398380 (hyl), MN398381 (gelE) and MN420464 (esp). These isolates were used as positive controls.

Results

Virulence phenotype

Of the 140 isolates, 58 (41.4%) (E. faecalis (n = 53), E. faecium (n = 5)) isolates were found to be beta-hemolytic. All the beta-hemolytic E. faecium isolates were those isolated from urine. Gelatinase was produced by 55 (39.3%) isolates (E. faecalis (n = 33), E. faecium (n = 22)). Slime production was detected in 130 (92.8%) isolates (E. faecalis (n = 82), E. faecium (n = 48)).

Virulence genotype

Among the 5 virulence determinants screened, asa1 were significantly more common in E. faecalis followed by gelE, while, gelE was the most common gene followed by esp in E. faecium. cylA, gelE and asa1 were significantly more common in E. faecalis (59.55%, 85.39%, 86.51%) than E. faecium (1.96%, 60.78%, 9.80%) (p < 0.0001, p = 0.001967, p < 0.0001). hyl was detected only in E. faecium (5.9%) and not in E. faecalis (0%) (p = 0.0465). However, no difference was observed in the incidence of esp between species ((E. faecalis (53.93%) vs. E. faecium (45.09%), p = 0.406164).

Correlation between virulence phenotype and genotype

Among the 53 beta-hemolytic E. faecalis isolates, 48 (90.6%) harboured the cylA gene. Among the E. faecalis isolates, 33/33 (100%) gelatinase producers and 43/56 (77.8%) non-gelatinase producers harboured gelE. Among E. faecium isolates, 14/22 (63.6%) gelatinase producers and 17/29 (58.6%) non-gelatinase producers harboured the gelE. Among the E. faecalis (n = 82) isolates that were slime producers, 45 (54.9%) and 73 (89.02%) harboured esp and asa1 while, 3 (42.9%), 4 (57.1%) of the non-slime producers (n = 7) possessed esp and asa1 respectively. Among the E. faecium isolates, 22/48 (45.8%), 4/48 (8.3%) of the slime producers and 1/3 (33.3%), 1/3 (33.3%) of the non-slime producers harboured the genes, esp and asa1 respectively (Table 1).

Table 1 Correlation between virulence phenotype and genotype
Full size table

Virulence score

Majority of the E. faecalis isolates causing UTI elaborated VFs compared to E. faecium (p < 0.0001, OR = 163.3333, 95% CI 20.2715–1316.0268) (Table 2). The number of VFs detected in each isolate was recorded as the VF score. Majority of the E. faecalis isolates had a VF score 4 (34.83%), followed by score 3 (26.96%), score 2 (28.08%), score 1 (8.98%) and score 0 (1.12%). Nevertheless, the VF score pattern exhibited by E. faecium was found to be in the reverse order: VF score 4 (1.96%), followed by score 3 (7.84%), score 2 (25.49%), score 1 (41.18%) and score 0 (23.53%) (Table 2). VF score 4 and 3 were quite common among E. faecalis than E. faecium (p < 0.0001, 0.0124) respectively. Nevertheless, VF score 1 was significantly associated with E. faecium (< 0.0001) (Table 3).

Table 2 Correlation of the virulence score of the enterococci with clinical source
Full size table
Table 3 Comparison of VF scores between E. faecalis vs E. faecium
Full size table

Discussion

In our study, 39.3% of enterococci (E. faecalis (37.1%), E. faecium (43.1%)) were gelatinase producers. Our results are in concordance with previous Indian studies that have documented a lower incidence of gelatinase production in Enterococci [26, 27]. Recent studies have reported an incidence of gelE in the range of 31–91.4% (Additional file 1: Table S1). Our molecular studies indicated that gelE was the second most common (76.4%) VF detected in enterococci, more commonly in E. faecalis (85.39%) than E. faecium (60.78%). Among the E. faecalis studied, all the gelatinase producers (100%) harboured gelE gene while, the reverse was not true. In contrary, 63.6% of the gelatinase producing E. faecium isolates harboured gelE gene. In concordance with previous reports, gelE was present as a silent gene in E. faecalis (77.8%), and E. faecium (58.6%) [18, 28].

In line with previous reports, 41.43% of our enterococcal isolates were beta-hemolytic [26, 27, 29]. In our study, of the beta-hemolytic enterococci (41.43%), majority were E. faecalis (91.38%) while, only 8.62% were E. faecium isolates. Our results corroborate with previous reports that document a very low frequency of cylA among E. faecium compared to E. faecalis (Additional file 1: Table S1) Of note, all the beta-hemolytic E. faecium were urinary isolates that did not harbour the gene cylA. Nevertheless, majority (90.57%) of the beta-hemolytic E. faecalis (urine (71.7%), pus (16.98%), blood (1.89%)) harboured the cylA gene. This finding is of clinical significance as the frequency of death is five times higher in an enterococcal infection associated with cytolysin-producing strain compared to a non cytolysin-producing strain [30]. In our study, cylA was present as a silent gene in 13.88%, 2.17% of E. faecalis and E. faecium respectively.

Esp encoded by esp is associated with adhesion, colonisation and host immune evasion. Though previous reports suggest that esp is more common in E. faecium, in our study, incidence of esp was slightly higher in E. faecalis (53.93%) than E. faecium (45.09%) [31, 32]. The incidence of esp and asa1 shows a wide variation in various reports (Additional file 1: Table S1). Among the slime producers, 54.9%, 89.02% of E. faecalis isolates, and 45.8%, 8.3% of E. faecium harboured esp and asa1 respectively. In our study, esp and asa1 were found to be silent genes in both E. faecium (33.3%, 33.3%) and E. faecalis isolates (42.9%, 57.1%). As reported earlier, hyl was detected only in E. faecium [33]. Nevertheless, a few studies have reported the incidence of hyl in both species [29, 34,35,36,37,38,39]. Significant difference was observed in the VF score between species. In line with previous studies, E. faecalis (61.8%) were found to be multi-virulent with VF Scores 4 or 3 while, VF score 1 was quite common in E. faecium (41.18%) [27, 36]. Majority of the urinary E. faecalis elaborated multiple VFs compared to E. faecium.

Non-expression of these virulence genes could be attributed to a triad of factors, (i) gene expression is triggered in late exponential phase at high cell densities, (ii) environmental factors might influence gene expression and (iii) in vitro phenotypic testing conditions are different from the in vivo conditions [28, 40]. Nevertheless, the presence of virulence determinants in the clinical isolates might contribute to increased severity as they could be expressed under optimum conditions in vivo. Metadata of the previous studies on the detection of virulence genes of enterococci by multiplex/duplex/simplex PCR is depicted in Additional file 1: Table S1 [18, 27, 29, 33,34,35,36,37,38,39, 41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59].

Conclusion

We report that our simple modification of the existing multiplex PCR had increased the detection of the enterococcal virulence genes. Predominance of virulence genes was in order of gelE (76.43%) > asa1 (58.57%) > esp (50.71%) > cylA (38.57%) > hyl (2.14%). Virulence determinants were more common in E. faecalis (asa1 (86.51%), gelE (85.39%), cylA (59.55%)) than E. faecium (asa1 (9.80%), gelE (60.78%), cylA (1.96%)). hyl was detected only in E. faecium. This modified PCR protocol could be useful to resolve the problem of decreased detection of virulence determinants in enterococci.

Limitations of the study

This study lacks the analysis of other virulence factors elaborated by enterococci. Also, majority of the study isolates were from urine with very less number from other body fluids.

Availability of data and materials

GenBank Accession numbers: MN398378 (asa1), MN398379 (cylA), MN398380 (hyl), MN398381 (gelE) and MN420464 (esp).

Abbreviations

AS:

Aggregation substance

Cyl:

Cytolysin

Esp:

Enterococcal surface protein

Gel:

Gelatinase

Hyl:

Hyaluronidase

PCR:

Polymerase chain reaction

References

  1. Chow JW, Thal LA, Perri MB, Vazquez JA, Donabedian SM, Clewell DB, Zervos MJ. Plasmid-associated hemolysin and aggregation substance production contribute to virulence in experimental enterococcal endocarditis. Antimicrob Agents Chemother. 1993;37:2474–7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. 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.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Ike Y, Hashimoto H, Clewell DB. High incidence of hemolysin production by Enterococcus (Streptococcus) faecalis strains associated with human parenteral infections. J Clin Microbiol. 1987;25:1524–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Gilmore MS, Segarra RA, Booth MC. An HlyB-type function is required for expression of the Enterococcus faecalis hemolysin/bacteriocin. Infect Immun. 1990;58:3914–23.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Ike Y, Clewell DB, Segarra RA, Gilmore MS. Genetic analysis of the pAD1 hemolysin/bacteriocin determinant in Enterococcus faecalis: Tn917 insertional mutagenesis and cloning. J Bacteriol. 1990;172:155–63.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Su YA, Sulavik MC, He P, Makinen KK, Makinen PL, Fiedler S, Wirth R, Clewell DB. Nucleotide sequence of the gelatinase gene (gelE) from Enterococcus faecalis subsp liquefaciens. Infect Immun. 1991;59:415–20.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Gutschik E, Moller S, Christensen N. Experimental endocarditis in rabbits. Significance of the proteolytic capacity of the infecting strains of Streptococcus faecalis. Acta Pathol Microbiol Scand. 1979;87:353–62.

    CAS  Google Scholar 

  8. Coque TM, Steckelberg JM, Patterson JE, Murray BE. Possible virulence factors of enterococci. 1993; abstr. 1166. Program Abstr. 33rd Intersci. Conf. Antimicrob Agents Chemother. 

  9. Franz CMAP, Huch M, Abriouel H, Holzapfeland W, Gálvez A. Enterococci as probiotics and their implications in food safety. Int J Food Microbiol. 2011;151(2):125–40.

    Article  CAS  PubMed  Google Scholar 

  10. Del Papa MF, Hancock LE, Tomas VC, Perego M. Full activation of Enterococcus faecalis gelatinase by a C terminal proteolytic cleavage. J Bacteriol. 2007;189(24):8835–43.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  11. 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.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Shankar N, Lockatell CV, Baghdayan AS, Drachenberg C, Gilmore MS, Johnson DE. Role of Enterococcus faecalis surface protein, Esp in the pathogenesis of ascending urinary tract infection. Infect Immun. 2001;69:4366–72.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Toledo-Arana A, Valle J, Solano C, Arrizubieta MJ, Cucarella C, Lamata M, Amorena B, Leiva J, Penades JR, Lasa I. The enterococcal surface protein, Esp, is involved in Enterococcus faecalis biofilm formation. Appl Environ Microbiol. 2001;67:4538–45.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Galli D, Lottspeich F, Wirth R. Sequence analysis of Enterococcus faecalis aggregation substance encoded by the sex pheromone plasmid pAD1. Mol Microbiol. 1990;4:895–904.

    Article  CAS  PubMed  Google Scholar 

  15. Kreft B, Marre R, Schramm U, Wirth R. Aggregation substance of Enterococcus faecalis mediates adhesion to cultured renal tubular cells. Infect Immun. 1992;60:25–30.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Guzman CA, Pruzzo C, LiPira G, Calegari L. Role of adherence in pathogenesis of Enterococcus faecalis urinary tract infection and endocarditis. Infect Immun. 1989;57:1834–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Olmsted SB, Dunny GM, Erlandsen SL, Wells CL. A plasmid-encoded surface protein on Enterococcus faecalis augments its internalization by cultured intestinal epithelial cells. J Infect Dis. 1994;170:1549–56.

    Article  CAS  PubMed  Google Scholar 

  18. Vankerckhoven V, Van Autgaerden T, Vael C, Lammens C, Chapelle S, Rossi R, Jabes D, Goossens H. Development of a multiplex PCR for the detection of asa1, gelE, cylA, esp, and hyl genes in enterococci and survey for virulence determinants among European hospital isolates of Enterococcus faecium. J Clin Microbiol. 2004;42(10):4473–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Rice LB, Carias L, Rudin S, Vael C, Goossens H, Konstable C, Clare I, Nallapareddy SR, Huang W, Murray BE. A potential virulence gene, hylEfm, predominates in Enterococcus faecium of clinical origin. J Infect Dis. 2003;187:508–12.

    Article  CAS  PubMed  Google Scholar 

  20. Hynes WL, Walton SL. Hyaluronidases of gram-positive bacteria. FEMS Microbiol Lett. 2000;183:201–7.

    Article  CAS  PubMed  Google Scholar 

  21. Facklam RR, Collins MD. Identification of Enterococcus species isolated from human infections by a conventional test scheme. J Clin Microbiol. 1989;27(4):731–4.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Fernandes SC, Dhanashree B. Drug resistance & virulence determinants in clinical isolates of Enterococcus species. Indian J Med Res. 2013;137:981–5.

    PubMed  PubMed Central  Google Scholar 

  23. Freeman DJ, Falkiner FR, Keane CT. New method for detecting slime production by coagulase negative Staphylococci. J Clin Pathol. 1989;42(8):872–4.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Coque TM, Patterson JE, Steckelberg 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;171:1223–9.

    Article  CAS  PubMed  Google Scholar 

  25. Willems RJ, Homan W, Top J, Santen-Verheuvel M, Tribe D, Manzioros X, Gaillard C, Vandenbroucke-Grauls CM, Mascini EM, Van Kregten E, Van Embden JD, Bonten MJ. Variant esp gene as a marker of a distinct genetic lineage of vancomycin-resistant Enterococcus faecium spreading in hospitals. Lancet. 2001;357:853–5.

    Article  CAS  PubMed  Google Scholar 

  26. Ravichandran L, Sivaraman U, Pramodhini S, Srirangaraj S, Seetha KS. Prevalence of virulence factors among clinical isolates of Enterococcus spp. Asian J Pharm clin Res. 2016;9(9):72–5.

    Article  CAS  Google Scholar 

  27. Padmasini E, Divya G, Karkuzhali M, Padmaraj R, Srivani Ramesh S. Distribution of cylA, esp, asa1, hyl and gelE virulence genes among clinical isolates of Enterococcus faecium and Enterococcus faecalis. BMC Infect Dis. 2014;14(S3):P32.

    Article  PubMed Central  Google Scholar 

  28. Eaton TJ, Gasson MJ. Molecular screening of Enterococcus virulence determinants and potential for genetic exchange between food and medical isolates. Appl Environ Microbiol. 2001;67:1628–35.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Banerjee T, Anupurba S. Prevalence of virulence factors and drug resistance in clinical isolates of Enterococci: a study from North India. J Pathog. 2015;2015:692612.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  30. Huycke MM, Spiegel CA, Gilmore MS. Bacteremia caused by hemolytic, high-level gentamicin-resistant Enterococcus faecalis. Antimicrob Agents Chemother. 1991;35(8):1626–34.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Tendolkar PM, Baghdayan AS, Shankar N. Pathogenic enterococci: new developments in the 21st century. Cell Mol Life Sci. 2003;60(12):2622–36.

    Article  CAS  PubMed  Google Scholar 

  32. Araújo TF, Ferreira CLDLF. The genus enterococcus as probiotic: safety concerns. Braz Arch Biol Technol. 2013;56(3):457–66.

    Article  Google Scholar 

  33. Dogru AK, Gencay YE, Ayaz ND. Comparison of virulence gene profiles of Enterococcus faecium and Enterococcus faecalis chicken neck skin and faeces isolates. Kafkas Üniversitesi Veteriner Fakültesi Dergisi. 2010;16:129–33.

    Google Scholar 

  34. Biswas PP, Dey S, Sen A, Adhikari L. Molecular characterization of virulence genes in vancomycin-resistant and vancomycin-sensitive enterococci. J Glob Infect Dis. 2016;8(1):16–24.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Ferguson DM, Talavera GN, Hernández LA, Weisberg SB, Ambrose RF, Jay JA. Virulence Genes among Enterococcus faecalis and Enterococcus faecium isolated from coastal beaches and human and non-human sources in Southern California and Puerto Rico. J Pathog. 2016;2016:3437214.

    Article  PubMed  PubMed Central  Google Scholar 

  36. Strateva T, Atanasova D, Savov E, Petrova G, Mitov I. Incidence of virulence determinants in clinical Enterococcus faecalis and Enterococcus faecium isolates collected in Bulgaria. Braz J Infect Dis. 2016;20(2):127–33.

    Article  PubMed  Google Scholar 

  37. Heidari H, Hasanpour S, Ebrahim-Saraie HS, Motamedifar M. High incidence of virulence factors among clinical Enterococcus faecalis isolates in Southwestern Iran. Infect Chemother. 2017;49(1):51–6.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Shokoohizadeh L, Ekrami A, Labibzadeh M, Ali L, Alavi SM. Antimicrobial resistance patterns and virulence factors of enterococci isolates in hospitalized burn patients. BMC Res Notes. 2018;11:1.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  39. Golob M, Pate M, Kušar D, Dermota U, Avberšek J, Papic B, Zdovc I. Antimicrobial Resistance and virulence genes in Enterococcus faecium and Enterococcus faecalis from humans and retail red meat. BioMed Res Int. 2019;2019:2815279.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  40. Finlay BB, Falkow S. Common themes in microbial pathogenicity revisited. Microbiol Mol Biol Rev. 1997;61(2):136–69.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Billström H, Lund B, Sullivan A, Nord CE. Virulence and antimicrobial resistance in clinical Enterococcus faecium. Int J Antimicrob Agents. 2008;32(5):374–7.

    Article  PubMed  CAS  Google Scholar 

  42. Martín-Platero AM, Valdivia E, Maqueda M, Martínez-Bueno M. Characterization and safety evaluation of enterococci isolated from Spanish goats’ milk cheeses. Int J Food Microbiol. 2009;132:24–32.

    Article  PubMed  CAS  Google Scholar 

  43. Zoletti GO, Pereira EM, Schuenck RP, Teixiera LM, Siqueira JF Jr, dos Santos KR. Characterization of virulence factors and clonal diversity of Enterococcus faecalis isolates from treated dental root canals. Res Microbiol. 2011;162(2):151–8.

    Article  CAS  PubMed  Google Scholar 

  44. Ribeiro T, Oliveira M, Fraqueza MJ, Lauková A, Elias M, Tenreiro R, Barreto AS, Semedo-Lemsaddek T. Antibiotic resistance and virulence factors among Enterococci isolated from chouriÇo, a traditional Portuguese dry fermented sausage. J Food Prot. 2011;74(3):465–9.

    Article  CAS  PubMed  Google Scholar 

  45. Hasani A, Sharifi Y, Ghotaslou R, Naghili B, Hasani A, Aghazadeh M, Milani M, Bazmani A. Molecular screening of virulence genes in high-level gentamicin resistant Enterococcus faecalis and Enterococcus faecium isolated from clinical specimens in North west Iran. Indian J Med Microbiol. 2012;30(2):175–81.

    Article  CAS  PubMed  Google Scholar 

  46. Medeiros AW, Pereira RI, Oliveira DV, Martins PD, d’Azevedo PA, Van der Sand S, Frazzon J, Frazzon AP. Molecular detection of virulence factors among food and clinical Enterococcus faecalis strains in South Brazil. Braz J Microbiol. 2014;45(1):327–32.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Abdeen EE, Hussien H, Hussan Z, Abdella W. Genotyping and virulence genes of Enterococcus faecalis Isolated form Kareish cheese and minced meat in Egypt. Res J Microbiol. 2016;11:133–8.

    Article  CAS  Google Scholar 

  48. Tuhina B, Anupurba S, Karuna T. Emergence of antimicrobial resistance and virulence factors among the unusual species of enterococci, from North India. Indian J Pathol Microbiol. 2016;59:50–5.

    PubMed  Google Scholar 

  49. Wu X, Hou S, Zhang Q, Ma Y, Zhang Y, Kan W, Zhao X. Prevalence of virulence and resistance to antibiotics in pathogenic enterococci isolated from mastitic cows. J Vet Med Sci. 2016;78(11):1663–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Zheng JX, Wu Y, Lin ZW, Pu ZY, Yao WM, Chen Z, Li DY, Deng QW, Qu D, Yu ZJ. Characteristics of and virulence factors associated with biofilm formation in clinical Enterococcus faecalis isolates in China. Front Microbiol. 2017;24(8):2338.

    Article  Google Scholar 

  51. Kashef M, Alvandi A, Hasanvand B, Azizi M, Abiri R. Virulence factor and biofilm formation in clinical enterococcal isolates of the West of Iran. Jundishapur J Microbiol. 2017;10(7):e14379.

    Article  CAS  Google Scholar 

  52. Yang F, Zhang S, Shang X, Wang X, Yan Z, Li H, Li J. Short communication: antimicrobial resistance and virulence genes of Enterococcus faecalis isolated from subclinical bovine mastitis cases in China. J Dairy Sci. 2019;102(1):140–4.

    Article  CAS  PubMed  Google Scholar 

  53. Pillay S, Zishiri OT, Adeleke MA. Prevalence of virulence genes in Enterococcus species isolated from companion animals and livestock. Onderstepoort J Vet Res. 2018;85(1):e1–8.

    Article  PubMed  Google Scholar 

  54. Song H, Bae Y, Jeon E, Kwon Y, Joh S. Multiplex PCR analysis of virulence genes and their influence on antibiotic resistance in Enterococcus spp. isolated from broiler chicken. J Vet Sci. 2019;20(3):e26.

    Article  PubMed  PubMed Central  Google Scholar 

  55. Aladarose BE, Said HS, Abdelmegeed ES. Incidence of virulence determinants among enterococcal clinical isolates in egypt and its association with biofilm formation. Microb Drug Resist. 2019;6:880–9.

    Article  CAS  Google Scholar 

  56. Haghi F, Lohrasbi V, Zeighami H. High incidence of virulence determinants, aminoglycoside and vancomycin resistance in enterococci isolated from hospitalized patients in Northwest Iran. BMC Infect Dis. 2019;19(1):744.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  57. Shridhar S, Dhanashree B. Antibiotic Susceptibility Pattern and Biofilm Formation in Clinical Isolates of Enterococcus spp. Interdiscip Perspect Infect Dis. 2019;3:7854968.

    Google Scholar 

  58. Mohanty S, Behera B, Praharaj AK. Identification, antimicrobial susceptibility and virulence factors of Enterococcus species isolated from clinical specimens at an Indian tertiary care hospital. Int J Infect Dis. 2019;79(S1):49.

    Article  Google Scholar 

  59. Stępień-Pyśniak D, Hauschild T, Kosikowska U, Dec M, Urban-Chmiel R. Biofilm formation capacity and presence of virulence factors among commensal Enterococcus spp. from wild birds. Sci Rep. 2019;9(1):11204.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

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Acknowledgements

We would like to thank the Department of Science & Technology, India for the instrumentation facility provided through Fund for Improvement of Science & Technology (FIST) (SR/FST/College -2017/23).

Funding

This research work was not funded by any organisation.

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Authors and Affiliations

  1. Department of Microbiology, Research Laboratory for Oral and Systemic Health, Sree Balaji Dental College and Hospital, BIHER, Velachery Main Road, Chennai, 600100, India

    Alexander Kiruthiga & Kesavaram Padmavathy

  2. Department of Microbiology, Priyadarshini Dental College and Hospital, Pandur, Thiruvallur, India

    Alexander Kiruthiga

  3. Department of Laboratory Medicine, Meitra Hospital, Calicut, India

    Praveen Shabana

  4. ImmuGenix Biosciences Pvt Ltd, Chennai, India

    Venkatesan Naveenkumar

  5. Department of Microbiology, Sri Muthukumaran Medical College Hospital and Research Institute, Chennai, India

    Sumathi Gnanadesikan & Jeevan Malaiyan

Authors
  1. Alexander Kiruthiga
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  2. Kesavaram Padmavathy
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  3. Praveen Shabana
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  4. Venkatesan Naveenkumar
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  6. Jeevan Malaiyan
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Contributions

Conceptualisation: KP and VN, Experimentation: AK, KP, Supervision: VN, PS, Sample collection: SG, JM, Writing original draft: AK, KP. Review and editing: VN, PS, SG, JM. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Kesavaram Padmavathy.

Ethics declarations

Ethics approval and consent to participate

This research work has been reviewed and approved by the Institutional Ethical Committee, Sree Balaji Dental College & Hospital, BIHER, Chennai, India (IEC No: SBDCECM106/14/08/dt19.06.2014).

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Not applicable.

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The authors declare they have no competing interests.

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Kiruthiga, A., Padmavathy, K., Shabana, P. et al. Improved detection of esp, hyl, asa1, gelE, cylA virulence genes among clinical isolates of Enterococci. BMC Res Notes 13, 170 (2020). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13104-020-05018-0

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BMC Research Notes

ISSN: 1756-0500

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