Effects of Osmotic Stress and Sodium Hypochlorite on Endodontic Microbiota: An In-Vitro Study

OBJECTIVE: To assess the effect of osmotic stress on various bacteria in a planktonic milieu and the effect of exposure to sodium hypochlorite (NaOCl) on the microbial cells previously subjected to osmotic stress. METHODS: Enterococcus faecalis, Streptococcus sanguinis, Fusobacterium nucleatum, Porphyromonas gingivalis and Prevotella intermedia were suspended as follows: Iso-osmotic group 0.9% NaCl; Hypo-osmotic group “ultrapure water”; Hyper-osmotic group 9% NaCl solution for 120 hours before exposure to 0.0001% NaOCl for 10 minutes. Quantitative analyses of viable cells were performed at 0 and 120 hours and after exposure to NaOCl to obtain colony forming units (CFU/mL). A linear mixed-effects model was used to find the association between mean CFU/mL (logarithmic transformation) and the interaction of solution Group and Time (P<0.001). RESULTS: F. nucleatum, P. gingivalis and P. intermedia did not survive after 24 hours in any of the solutions and were excluded from further testing. For S. sanguinis there were significant differences at each time interval, when holding solution group constant. After 120 hours, the Hyper-osmotic group presented with the highest CFU/mL and was significantly different to the Iso-osmotic group (P<0.001). For E. Faecalis, there was a significant difference for each pairwise comparison of time (P<0.001) in mean CFU/mL between 0 hours and 120 hours for the Iso-osmotic and Hyper-osmotic groups. At 120 hours, no significant differences were found between the three groups. Significant differences were also found between 0 hours and Post-NaOCl administration, and between 120 hours and Post-NaOCl administration for all three groups (P<0.001). Exposure to NaOCl after hypo-osmotic stress was associated with significantly less CFU/mL for S. sanguinis compared to hyperosmosis and iso-osmosis (P<0.001) and for E. Faecalis only compared to hyperosmosis (P<0.001). CONCLUSION: S. sanguinis and E. faecalis were able to withstand osmotic stress for 120 hours. Hypo-osmotic stress before contact with NaOCl was associated with lower viable bacterial numbers, when compared to the other media for the above species. Hyper-osmotic stress was associated with higher viable bacterial numbers after NaOCl exposure for E. faecalis.