Prevalence and virulence factors of haemolytic Enterococcus faecalis isolated from root filled teeth associated with periradicular lesions: A laboratory investigation in Thailand

Aim: Previous endodontic research has provided limited understanding of the prevalence and roles of haemolytic and non-haemolytic Enterococcus faecalis strains in root filled teeth. This study aimed to determine the prevalence of these strains in root filled teeth with periradicular lesions and investigate their associated virulence factors. Methodology: A total of 36 root canal samples were collected from 36 subjects. The prevalence of E. faecalis was determined using culture and PCR methods. Antibiotic susceptibility of haemolytic and non-haemolytic E. faecalis strains was assessed using the broth dilution assay. The cytokine stimulation in periodontal ligament (PDL) cells and neutrophil migration were evaluated using real-time PCR and migration assay, respectively. Cell invasion ability of the strains was assessed using a cell culture model. Additionally, the virulence gene expression of the haemolytic and non-haemolytic strains was investigated using real-time PCR. The Mann-Whitney U and Spearman's rho tests were used to examine the significant difference between the two strains and to analyse the correlation between phenotype and gene expression, respectively. Results: Enterococcus faecalis was detected in 33.3% and 88.9% of samples by culture and real-time PCR, respectively. Haemolytic strains were found in 36.4% of subjects. Non-haemolytic strains exhibited susceptibility to erythromycin and varying susceptibility to tetracycline, while all haemolytic strains were resistant to both antibiotics. Haemolytic strains significantly upregulated the expression of IL-8, OPG and RANKL in PDL cells (p < .05). Notably, the fold increases in these genes were higher: IL-8 (556.1 +/- 82.9 vs. 249.6 +/- 81.8), OPG (2.2 +/- 0.5 vs. 1.3 +/- 0.2) and RANKL (1.8 +/- 0.3 vs. 1.2 +/- 0.1). Furthermore, haemolytic strains had a greater effect on neutrophil migration (68.7 +/- 15.2% vs. 46.9 +/- 11.4%) and demonstrated a higher level of internalization into oral keratinocyte cells (68.6 +/- 0.4% vs. 33.8 +/- 0.5%) (p < .05). They also showed enhanced expression of virulence genes associated with haemolysin, surface proteins, collagen-binding and aggregation substances. Gelatinase activity was only detectable in non-haemolytic strains. Conclusions: This study revealed that haemolytic strains E. faecalis possessed enhanced abilities in host invasion and a higher abundance of virulence factors, suggesting their potential contribution to more severe disease manifestations.