Besides B. sporothermodurans, a few other spore-forming species have also been reported to sporadically contaminate UHT- or sterilized milk with direct or indirect evidence for the production of highly heat-resistant spores: Geobacillus (formerly Bacillus) stearothermophilus (see references in Rombaut et al. 2002), Br. brevis and/or Brevibacillus borstelensis (de Silva et al. 1998; Rombaut et al. 2002), B. sphaericus, B. licheniformis and Br. brevis (Cosentino et al. 1997) and Paenibacillus lactis (Scheldeman et al. 2004a). The production of spores with high resistance at 120–121°C is well known for G. stearothermophilus (Huemer et al. 1998; Brown 2000), but as this organism is a thermophile, spoilage problems may only occur in packs held at elevated temperatures. Brevibacillus brevis has been shown to produce spores heat resistant at 130°C (Rombaut et al. 2002). The best documented recent event is a periodical but tenacious contamination of UHT milk packages from one dairy plant with spores of P. lactis. Indirect evidence that this new species, described by Scheldeman et al. (2004a), can produce highly heat-resistant spores comes from the fact that the contaminated UHT milk packages came from different processing lines (direct and indirect UHT) and were co-contaminated with B. sporothermodurans. Interestingly, P. lactis strains had also been isolated on different dairy farms from raw milk, milking apparatus and filter cloth. One of these farm strains, isolated from a cluster of the milking apparatus, showed the same REP-PCR patterns as the UHT isolates. In this case, there seems to be for the first time plausible evidence of a direct link of contamination with highly heat-resistant spores from the raw milk on the dairy farm to the heat-treated milk in the dairy.
Only a few studies have addressed the presence of highly heat-resistant spores at the dairy farm (Table 2), which can be the original source for spores causing spoilage, poisoning or contamination of heat-treated milk upon germination and growth in the final product with long shelf-life. The most comprehensive study of potentially highly heat-resistant spore formers was performed recently by Scheldeman et al. (2005) investigating raw milk, milking equipment after the heat-cleaning procedure (teat cups, clusters, connection points, filter cloth, collection tank), green crop (silage, maize, hay/straw) and fodder (feed concentrate, pulp, cereals) in the winter period at 17 dairy farms in geographically different locations in Belgium. The notation ‘potentially highly heat-resistant spore formers’ was used because the selective heat treatment of the samples (30 min at 100°C) could not only select for spores with a high intrinsic heat resistance but also for spores with a lower heat resistance which were very abundant in the sample. Depending on the incubation temperature, high average counts of potentially highly heat-resistant spore formers were found in filter cloths (102–103 CFU g−1 at 37 and 55°C), green maize (102–103 CFU g−1 at 37°C), hay/straw (>103 CFU g−1 at 37°C) and feed concentrate samples (102–103 CFU g−1 at 20, 37 and 55°C). It is noteworthy that 10–20% of the concentrate and self-made mixtures contained >102 CFU g−1 of psychrotolerant spore formers, which are of main interest in the cold milk chain. After a polyphasic taxonomic characterization of the potentially highly heat-resistant spore-forming isolates, a very large taxonomic diversity was found covering as much as seven genera (Aneurinibacillus, Bacillus, Brevibacillus, Geobacillus, Paenibacillus, Ureibacillus and Virgibacillus). In Fig. 2 a neighbour-joining tree is shown to reflect the phylogenetic diversity of these spore formers. Eighteen previously unknown taxa were found, covering 23% of all isolates, of which five have been described as a result of this study: Bacillus farraginis, Bacillus fortis and Bacillus fordii (Scheldeman et al. 2004b), P. lactis (Scheldeman et al. 2004a) and Bacillus ruris (Heyndrickx et al. 2005). Overall, B. licheniformis and the thermophile Geobacillus (formerly Bacillus) pallidus were the most frequently isolated species. Besides these two species, also B. farraginis and members of the Bacillus subtilis group were the most widely spread species across the sampled farms. In raw milk, 20 different species were found, of which B. licheniformis far outnumbered the other species. All investigated samples, and especially feed concentrate, hay and straw, silage, teat cups, clusters and filter cloth, were identified as possible entry points for potentially highly heat-resistant spores into raw milk. Sixty per cent of the different species found in raw milk were also recovered from feed concentrate, but some species such as P. lactis and Ureibacillus thermosphaericus, were only recovered from the milking equipment. Nevertheless, for five species, other entry points must exist as they were only found in raw milk in the course of this study.