Morphological Characterisation of Bacillus sporothermodurans Spores Using Various Microscopic Techniques

Introduction: Bacillus sporothermodurans are gram positive, mesophilic sporeformers that are able to produce highly-heat resistant spores (HRS). Information on the nature and origin of these spores may contribute to reduction in contamination and therefore improvement in the quality of dairy products. (GTG)₅ fingerprinting is known to be a promising genotypic tool that allows for more rapid and reliable results and has not yet been used in strain identification of B. sporothermodurans.

Purpose: In this study, (GTG)₅ fingerprinting was evaluated for B. sporothermodurans. In addition, the isolation of various strains from UHT milk as well as from the dairy farm were compared using REP PCR and (GTG)₅ PCR.

Methods: In this report, a collection of 9 B. sporothermodurans strains were obtained from UHT milk or farm sources (i.e., feed concentrate, silage and raw milk) from different countries. These strains were identified using the general B. sporothermodurans PCR as well as the more specific HRS-PCR method identifying the HRS-clone. REP PCR and (GTG)₅ PCR fingerprinting were performed on all 9 isolates and thereafter analyzed using a dendrogram.

Results: The two new UHT milk strains (i.e., QA1 from Belgium and F3 from South Africa) were positive for the HRS-PCR. Four of the 9 isolates from raw milk or feed concentrate were tested negative for the HRS-PCR. The farm strain MB 1505 sourced from silage was positive for the HRS-PCR. From the dendrogram, B. sporothermodurans strains showed an overall similarity of 40%. The three strains (QA1, F3 and MB 372) showed slight differences in similarity to the other strains. This would be expected as all the three strains were isolated form UHT milk. It was observed that even though the feed concentrate strains all originated from Belgium, the similarity between them was not more than 80% in both molecular methods.

Significance: (GTG)₅ PCR has been used to produce DNA fingerprints for a number of species and in this present study, we showed that this method could be used to fingerprint B. sporothermodurans strains.