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

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 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].  [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.
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.

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%)  [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    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.

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.