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. The specimen sources of hospital-acquired isolates included urinary tract infection (UTI) (78, 88.6%), wound (7, 7.9%) and blood (3, 3.4%). The specimens were obtained from different wards including outpatients (35, 39.8%), intensive (23, 26.1%), intensive care units (ICU) (12, 13.6%), infectious ward (13, 14.8%), emergency ward (3, 3.4%), ear-nose-throat (ENT) (1, 1.1%), urology and nephrology (1, 1.1%). 42 (47.7%) isolates were from male and 46 (52.3%) were from female cases. The age range of patients was from 2 months to 86 years, with a mean of 39.04 years. 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. 49 (67.1%) of the isolates were obtained from the males and 24 (32.9%) from the females. The age range of endodontic treatment patients was 12-66 years, with a mean of 32.41 years. Briefly to collect the isolates, 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. Root canal of teeth with no prior endodontic treatment and teeth with previously root canal treatment that showed secondary infection was removed by drill and endodontic K-files without using any chemical solvents. After sampling the single root canal and multi-root canal of the teeth, 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.
Results
All isolates were investigated for the biofilm formation, in which the minimum, maximum and average of biofilm formation (OD570 nm) were 0.054, 2.325 and 1.611, respectively. Most isolates showed strong biofilm formation (94, 58.4%), while 10 (6.2%) displayed no biofilm formation. Biofilm formation of hospital-acquired isolates was significantly higher than the dental root canal isolates (P = 0.023). Most of the isolates showed no gelatinase activity (70.8%), while hospital-acquired isolates significantly displayed the most gelatinase activity (P = 0.001). In addition, most isolates showed no hemolysis activity (80.7%), and all hemolysis activity was found in hospital-acquired isolates (19.2%). The most presence of the virulence genes among isolates were ace and efaA genes (88.8% and 85.1%, respectively), and the lowest one belonged to cylA and asa1 (7.5% and 14.9%, respectively). The presence of gelE (contributing to gelatinase activity) and cylA (contributing to hemolysis activity) was significantly associated with phenotype gelatinase and hemolysis activity, respectively (P < 0.001, P = 0.013). In addition, the presence of efaA, cylA, and gelE was significantly more in hospital-acquired isolates, as compared to dental-root canal (P = 0.002, P <0.001, P = 0.008, respectively). By comparing the presence of virulence genes among isolates, it was found that hospital-acquired isolates had higher virulence genes than dental root canal isolates (P = 0.007), such that all isolates had at least one virulence gene. The number of virulence genes was 1 to 7 among hospital-acquired isolates and 1 to 6 in the case of dental root canal isolates. Among hospital-acquired isolates, the presence of 5 and 4 virulence genes was the highest (36.4% and 30.7%, respectively); also, the presence of 4 and 3 virulence genes was the highest among isolates of the dental root canal (39.7% and 31.5%, respectively). In addition, efaA and gelE positive isolates had a higher biofilm formation than negative isolates in all isolates (P < 0.05). The mean of biofilm formation of the isolates with different virulence genes is presented in figure 1.
The occurrence of CRISPR-cas is shown in table 3. Overall, the presence of CRISPR1-cas loci in dental-root canal isolates (4 of 73) was lower than that of hospital-acquired isolates (17 of 88) (P = 0.008), whereas the presence of CRISPR3-cas in dental-root canal isolates (26 of 73) was higher than that of hospital-acquired isolates (2 of 88) (P < 0.001); also, orphan CRISPR2 made no difference between hospital-acquired and dental-root canal isolates. None of the isolates had, however, both of CRISPR1-cas and CRISPR3-cas, as well as CRISPR1-cas, orphan CRISPR2, and CRISPR3-cas, at the same time. The isolates were more likely to harbor orphan CRISPR2 than CRISPR1-cas and CRISPR3-cas. In addition, the presence of orphan CRISPR2 was significantly correlated with CRISPR1-cas (P = 0.031), whereas it was not significant with CRISPR3-cas. At least one CRISPR-cas locus was found in 106 (65.8%) of all isolates. The results, therefore, showed the isolates containing high virulence genes tended to have more frequently investigated cas genes. Overall, the absence of CRISPR1-cas in association with one of CRISPR1 or CRISPR3 was significantly correlated with the absence of the esp gene (P = 0.005, P = 0.033). In addition, the presence of either CRISPR1-cas or orphan CRISPR2 and either CRISPR3-cas or orphan CRISPR2 was significantly correlated with the presence of ace and the absence of gelE, respectively (P = 0.019, P =0.014). Other significant correlations were found between the absence of CRISPR1 and the absence of cylA, hyl, gelE, asa1 (P < 0.05), and between the absence of CRISPR2 and the absence of gelE (P = 0.001). In hospital-acquired isolates, a significant correlation was found between the absence of CRISPR loci and the absence of gelE, asa1, gelatinase and hemolysis activity (P < 0.05); in dental-root canal isolates, a significant correlation was found between the absence of CRISPR3-cas and the absence of gelatinase (P = 0.003), between the absence of either CRISPR1-cas or CRISPR2-cas and the absence of gelE (P = 0.021), and between the presence of orphan CRISPR2 and either orphan CRISPR2 or CRISPR3-cas and biofilm production (P = 0.046, P = 0.044, respectively) (see table 4).