Comparison of heating block and water bath methods to determine heat resistance in Shiga-toxin producing Escherichia coli with and without the locus of heat resistance

Abstract

This study found variability in the time required for tubes of media in heating block wells to reach 60 °C, resulting in significant effects on heat resistance measurements. To determine the extent that methodology changed heat resistance measurements, we compared the heat resistance of Shiga-toxin producing Escherichia coli (STEC) strains with and without the locus of heat resistance (LHR) using both heating block and water bath methods. A total of 34 strains of STEC were used along with a generic E. coli which has been identified as heat-resistant and used as a positive control. The E. coli strains were incubated in a water bath and a heating block set at 60 °C to determine come up time to 60 °C (T0) and for 6 additional minutes (T6) to calculate the D₆₀ value. After incubation, the colony forming units (CFU) were enumerated and mean log CFU/mL from biological replicates was calculated. To compare reductions from T0 to T6, standard deviations among replicates within heating method and correlation of the D₆₀ values generated across methods were determined using Mixed model and Correlation analyses. Our findings indicate that the method chosen to evaluate heat resistance of E. coli can dramatically influence results as there was not a significant correlation between D₆₀ values for the same isolate determined by water bath and heating block methods. The water bath method generates more reliable and consistent heat resistance data and should be used in future evaluations of heat resistance in E. coli. Moreover, PCR screening for the LHR would only be moderately useful for predicting phenotypic heat-resistance of E. coli. Considering water bath data only, LHR-positive STEC isolates were either moderately heat-resistant (1 to 5 log reduction) or heat-sensitive (> 5 log reduction). As LHR-negative STEC were also moderately heat-resistant, prediction of phenotypic heat resistance from genotype requires further refinement.

Keywords: Escherichia coli; Heating block; Locus of heat resistance; Thermal inactivation; Water bath.