Phenotypic and Genotypic characterisation of Bacillus sporothermodurans

Abstract

Ultra high temperature (UHT) method is a thermal heat treatment of milk that is able to destroy non- sporeformers and sporeformers. The UHT process uses a temperature of 135-140°C for 1-2 seconds. Bacillus sporothermodurans is the only known species whose spores are able to survive the UHT treatment. With companies having the disadvantage of not being able to increase the temperatures during milk processing as a result of negative sensory properties, a better understanding of the structure of B. sporothermodurans spores and how exactly B. sporothermodurans is able to survive such high temperatures has become of great importance in the dairy industry. The first objective of this study was to determine whether there was a genotypic difference in the structure of B. sporothermodurans spores between those strains isolated from UHT strains and those from other sources (i.e. raw milk, feed concentrate and silage). Due to the fact that B. sporothermodurans spores are becoming more widespread, other genetic tools were used to help confirm the presence of these strains and whether they have the heat resistant clonal gene, thereby confirming whether the spores are heat resistant (HRS) or not. Two recently isolated strains (i.e. F3 isolated from UHT milk in South Africa and QA1 isolated from UHT milk in Belgium) were identified using the general B. sporothermodurans method (BSPO PCR) and the more specific HRS-PCR method identifying the HRS clone. Thereafter the (GTG)5 PCR method was used to compare 9 B. sporothermodurans strains previously obtained from UHT milk or farm sources in different countries. Results showed that the two recently isolated UHT strains, QA1 and F3, had a close association shown by the (GTG)5 PCR patterns and were positive for the HRS clone. Results go on to show that the B. sporothermodurans strains of the HRS clone look to be primarily responsible for the production of heat resistant spores and that (GTG)5 PCR is a method that can be used to evaluate the genotypic differentiation of B. sporothermodurans strains. The second objective was to determine the effect of heat on the structure of Bacillus sporothermodurans spores. The spores of three B. sporothermodurans strains namely F3 (HRS strain isolated from UHT milk in South Africa), DSMZ 10599 (one of the first HRS strains isolated from UHT milk in Italy) and MB 1499 (a non HRS strain isolated from a feed concentrate in Belgium) were compared using various microscopic techniques. When observing the layers, an exosporial layer was present in all three strains. The second layer, the coat, showed to have multiple layers making up the spore coat. The first layer was the outer coat layer; the middle coat was made up of 4-5 lamellae layers and the last layer was the inner spore coat layer. A short, thick appendage like structure was observed in F3 developing from the middle coat layer. Differential scanning calorimetry was conducted on the B. sporothermodurans strain DSMZ 10599. The initial scan (thermogram from DSC) of DSMZ 10599 showed 3 transitions, one at 79°C, the second at 87°C and the third at 113°C. The initial peak was associated with the activation of the spores due to the heat. The other two endothermic peaks were most likely due to protein denaturation of the spore coat or cortex, or DNA unfolding in the spore core. The heat values were found to increase from the first to the last transition. UHT milk is heated to temperatures of up to 140°C and the DSC profile showed that spore death of strain DSMZ 10599 did not occur at this high temperature. The third objective was to determine the effects of H2O2 on the structure of B. sporothermodurans spores. The strains F3, DSMZ 10599 and MB 1668 were compared in terms of susceptibility to hydrogen peroxide. After 3 minutes, the exosporial and coat layer were completely degraded with significant swelling of the spore core. Rupture of the core of strains DSMZ 10599 and F3 occurred at 6 minutes. At 9 minutes, the core outer layer of F3 and MB 1668 was completely degraded however, not fully for DSMZ 10599. At 15 minutes the core was exposed to the environment however even though DSMZ 10599 core layer was still not completely degraded, the core was still exposed to the environment. It was concluded that (GTG)5 PCR is a method that can be used to evaluate the genotypic differentiation of B. sporothermodurans strains. When observing B. sporothermodurans phenotypically, for the first time, short appendage like structures were present protruding from the spore coat which may explain attachment to steel pipe surface resulting in post and cross contamination in UHT milk. A further investigation would be interesting to determine whether this species is able to produce a biofilm. It was also concluded that B. sporothermodurans spore structures do contribute to the resistance of heat and chemicals. It would be interesting to determine whether the damage done to the spore by heat is reversible.