Bacterial strains
This study was approved by the Regional Ethics Committee of Tabriz (Tabriz University of Medical Sciences, Tabriz, Iran, No. IR.TBZMED.REC.1397.188). A total of 88 isolates of E. faecalis were collected from Emam Reza Teaching and Treatment Hospital and pediatric hospitals of Tabriz, Iran. At the same time, in order to collect 73 dental-root canal isolates of E. faecalis, patients in need of endodontic treatment were referred to the clinic of the Faculty of Dentistry at Tabriz University of Medical Sciences, Tabriz, Iran. Briefly, after stages of access cavity preparation by the dentist, tooth and its surroundings were washed by sterile saline solutions and disinfected with 30% hydrogen peroxide followed by 2.5% sodium hypochlorite. Preexisting root canal filling was removed by drill and endodontic K-files without using any chemical solvents. After sampling the single-multi-rooted canal of the tooth, paper points were transferred to a tube containing Enterococcal broth (Becton Dickenson microbiology systems, Cockeysville, MD) and cultured on a bile esculin azide agar (Himedia, India) and incubated at 37 ºC for 24-48h [8]. Suspected colony was identified by the standard procedures of microbiology [33, 34] and genotype detection was performed by ddlE primer [35, 36], as shown in table 1. Both clinical and tooth identified isolates for further studies were stored in a trypticase soy broth containing 10% glycerol at -70 ºC.
Biofilm formation
Assessment of biofilm formation was done by quantitative biofilm formation in 96-well flat bottom polystyrene microplates under static conditions for 48h, as previously described [37, 38]. Briefly, for each isolate, a fresh colony cultured on a Muller-Hinton agar (Merck, Germany) containing 1% glucose was suspended in sterile saline and adjusted to 0.5 McFarland. 20 µl of the adjusted isolates was cultured in a 180 µl trypticase soy broth containing 1% glucose. After incubation for 48h at 37 ºC, each well was washed by the 1X phosphate buffer saline (PBS; pH 7.4), fixed by methanol, and stained by 200 µl 0.1% crystal violet for 30 min at room temperature. The excess crystal violet was discarded and washed by water flow. Biofilm formation was measured by the absorbance of the supernatant after being solubilized in 33% acetic acid at 570 nm by using a microtiter plate reader (BioTeck, Winooski, USA). The biofilm formation of each isolate was tested in three independent 96-well microplates and the average of three optical densities (OD) was used as the final biofilm formation value. The cut-off absorbance for biofilm formation was considered higher than OD = 0.524, which was the absorbance of the biofilm produced by E. faecalis ATCC® 29212™. The mean of the Biofilm formation of each isolate was grouped based on their level of distribution (OD570nm values) and categorized in quartiles higher than the cut-off absorbance and lower than the highest absorbance. Isolates whose absorbance of OD570nm fell below 0.524 were classed as non-biofilm formation, while those with 0.525-1.087 and 1.088-1.650 were grouped as low and moderate biofilm formation, respectively. Isolates with a biofilm formation greater than 1.651 were also considered with high biofilm formation.
Gelatinase production and hemolysis test
Hemolysis activity was assessed by blood agar plates prepared by a brain-heart infusion agar (BHI, biomerieux, Poland, Ltd) containing 5% of the group O Rh+ human blood. Cleared or green zone around the colonies was defined as hemolysis following incubation for 24h at 37 ºC [39].
Production of gelatinase was assessed by the degradation of gelatin on the X-ray radiographic film, as described by Pickett et al. [40]. The heavy inoculum of individual isolates was cultured in the tubes containing 3 ml MHB and a strip of the X-ray radiographic film which had been cut into small strips (approximately 6 by 30 mm). The tubes were incubated for 24h at 37 ºC and the cleared strip was defined as the production of gelatinase.
Genotype detection of virulence and cas genes
Total DNA for each isolate was extracted by the tissue buffer boiling method. Briefly, 20 µl tissue buffer (0.25% sodium doedecyl sulfate (SDS) and 0.05 M NaOH) were mixed with one colony of bacterial isolate and incubated at 95 ºC for 10 min. The suspension was centrifuged at 13000g for one minute and 180 µl DNase free water was added. Genotype analysis for each isolate was accomplished based on the multiplex polymerase chain reaction (PCR) of virulence determinants encoding the cytolysin activator cylA, hyl, esp, gelE, efaA, asa1, ace, ebpR, CRISPR1-cas, CRISPR1-cas csn1, CRISPR2, CRISPR3-cas and CRISPR3-cas csn1. Each of the primer sequences and the amplified size are shown in table 1. 2 µl of total DNA was used for the multiplex PCR in a 25µl reaction mixture. The mix for the detection of esp, cyl, hyl genes contained 12.5 µl of the PCR master mix (Yekta Tajhiz Azma, Iran), with 0.5 µM of each primer. The mix for ebp, asa1 and efaA had the same condition. The mix for the detection of gelE and ace contained 12.5 µl of the PCR master mix (Yekta Tajhiz Azma, Iran), 1.5 mM-additional MgCl2 and 0.5 µM of each primer. The mix for CRISPR1-cas csn1, CRISPR3-cas csn1 and CRISPR1-cas, CRISPR3-cas and CRISPR2 contained 12.5 µl of the PCR master mix (Yekta Tajhiz Azma, Iran), 1mM additional MgCl2, and 10 mM of each primer. The amplification condition was carried out with the following thermal cycling conditions: initial denaturation at 95 ºC for 10 min, 34 cycles of amplification consisting of 95 ºC for 30s, 30s at 58 ºC for esp, cylA, hyl, 58 ºC for efaA, 56 ºC for gel, ace, 52 ºC for ebpR, asa1, 60 ºC for all cas genes, and 72 ºC for 45s, with 72 ºC for 5 min in the final polymerization. PCR products were analyzed by electrophoresis in a 1% agarose gel at 100 V for 1 h in a 1X TBE buffer containing the DNA safe stain. PCR products size was correlated with a 100 based-pair DNA ladder (Yekta Tajhiz Azma, Iran) to confirm the conjunction with their expected PCR amplicon size. In addition, the PCR procedure for each isolate was carried out twice in the case of each primer in order to check the consistency and reproducibility.
Statistical analysis
SPSS software, version 17.0, (Chicago, IL, USA) was used for statistical analysis. T- test and one-tailed Fisher’s exact test were performed for data analysis. Significance was set at P ≤ 0.05.