Optimization of heat inactivation protocols for Orientia and Rickettsia species.

Heat treatment, or thermal disinfection, is one of the simplest and most widely used methods for microbial inactivation. Proper heat inactivation protocols are essential to ensure the safe transportation and handling of infectious materials, particularly for organisms in risk group 3, such as Rickettsia and Orientia. In this study, we examined the inactivation of four bacterial species-Orientia tsutsugamushi, Rickettsia typhi, Rickettsia conorii, and Rickettsia honei-at temperatures of 56 °C, 80 °C, and 90 °C for durations of 5, 15, 30, and 60 min. Observations were made at 0, 1, 3, 7, 10, and 14 days post-infection (dpi) to assess bacterial infectivity by monitoring bacterial DNA copies in newly infected cells. Our results indicate that 56 °C for 5 min was the minimum temperature and time required to inactivate O. tsutsugamushi, R. typhi, R. conorii, and R. honei. O. tsutsugamushi exhibited a higher reduction factor at 56 °C compared to R. typhi, R. conorii, and R. honei. Additionally, a strong inverse correlation between incubation time and log10 reduction factor was observed for O. tsutsugamushi and R. typhi, underscoring the importance of both time and temperature in effective heat treatment. However, no such correlation was observed for R. conorii and R. honei. These findings highlight the variable responses of bacteria to heat, emphasizing the need for pathogen-specific approaches in inactivation protocols. Optimizing heat treatment strategies based on these insights is critical for enhancing biosafety and ensuring effective pathogen eradication.