Thermal tolerance of E. faecalis to pulsed heating in the millisecond range

Abstract

Our aim was to evaluate thermal damage to endodontic pathogen Enterococcus faecalis (E. faecalis) caused by sub-second laser-generated heat pulses by determining the parameters for the thermal damage survival curve (TDSC). A novel experimental method for thermal pulsing of bacteria in the millisecond range was developed. After cultivation, E. faecalis was inoculated on anodized aluminum substrate and heated with a pulsed Nd:YAG laser. Viability was assessed with both plate count and flow cytometry methods. An E. faecalis TDSC for single-pulse millisecond range heating times was derived from the Arrhenius equation. Results gained from single-pulse heating viability measurements were used to predict the bactericidal effect of multiple sequential pulses (pulse train), and compared to experimental measurements. The thermal damage model was then applied to determine the relationship between laser fluence, pulse width, and the viability decrease of E. faecalis in a simulated root canal disinfection procedure. The application of the model to calculate the required lethal laser fluence levels on dentin during endodontic laser procedures seems to indicate that for endodontic procedures, the sub-millisecond pulsed Nd:YAG lasers are more effective in comparison with continuous-mode diode lasers and will cause less undesirable bulk heating of the tooth and surrounding tissues. The results of the study can be applied to create a model for predicting the impact of sub-second temperature increase on viability of bacteria on various surfaces and calculate required fluences and pulse widths to achieve the aforementioned effects with laser pulses.

Variable heat shock response model for medical laser procedures

“…This is supported by the observation that the plotted Fig. 1 Arrhenius curves according to published Arrhenius coefficients (full lines) [4,5,[10][11][12][13][14][15][16]18], together with imaginary Arrhenius curves representing the limiting damage processes at very short and very long exposure times (dotted lines), and the imaginary temperature versus exposure time curve (slashed line) representing the combined effect of both limiting processes. The publication references for the shown Arrhenius curves are in the legend listed from top to bottom corresponding to the curves starting from the steepest to the least steep Arrhenius curve.…”

“…This can be seen in Fig. 1, which shows the dependence of T crit on t exp as calculated for a number of published Arrhenius coefficients [4,5,[10][11][12][13][14][15][16][17][18].…”

“…Transient heating of tissues leading to protein denaturation and, consequently, to cellular stress or death is very common in medicine and biology, and it has been extensively studied, especially for exposure times longer than approximately 1 s [4][5][6][10][11][12][13][14][15]. Studies of cellular survival during shorter exposures to higher temperatures, for example, in a millisecond domain, which is very common during laser exposures, are less abundant and have been published more recently [16][17][18][19]. The published Arrhenius coefficients vary considerably, especially when comparing the results obtained at very short and very long exposures.…”

“…According to the VHS model, the critical temperature represents a combined effect of two limiting Arrhenius processes, defining cell viability at extremely long and short exposure times. The symbols represent the effective exposure times as calculated for the measured temperature pulse shapes and corresponding critical temperatures from [4,[16][17][18] by A 1 = (4.7 ± 1.4) × 10 89 s −1 and E 1 = (5.67 ± 0.11) × 10 7 J kmol −1 , and for the short exposure process 2 characterized by A 2 = (1.45 ± 0.15) × 10 4 s −1 and E 2 = (1.03 ± 0.03) × 10 7 J kmol −1 .…”

“…In the standard model originally proposed by Henriques and Moritz [4,6], based on measurements for t exp > 1 s, it is assumed that a single chemical process representing the kinetics of protein denaturation is involved and, therefore, the Arrhenius coefficients E and A are fixed and independent of the exposure time [10][11][12][13][14][15]. However, with more recent studies performed at extremely short exposure times (t exp < 10 ms), the obtained activation energies were significantly smaller and the damage threshold temperatures were significantly higher than what would be expected from the standard single-process Arrhenius model [16][17][18][19]. This finding is of particular significance in the context of medical laser procedures, where extremely short exposures are very common.…”