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Applied and Environmental Microbiology, March 2005, p. 1480-1494, Vol. 71, No. 3
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.3.1480-1494.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Incidence and Diversity of Potentially Highly Heat-Resistant Spores Isolated at Dairy Farms

Patsy Scheldeman,^1* Annelies Pil,^1,2, Lieve Herman,¹ Paul De Vos,² and Marc Heyndrickx¹

Department of Animal Product Quality, Centre for Agricultural Research, Ministry of the Flemish Community, Melle,¹ Laboratory of Microbiology (WE10), Faculty of Sciences, Ghent University, Ghent, Belgium²

Received 18 April 2004/ Accepted 11 October 2004

The presence of highly heat-resistant spores of Bacillus sporothermodurans in ultrahigh-temperature or sterilized consumer milk has emerged as an important item in the dairy industry. Their presence is considered undesirable since they hamper the achievement of commercial sterility requirements. By using a selective 30-min heat treatment at 100°C, 17 Belgian dairy farms were screened to evaluate the presence, sources, and nature of potentially highly heat-resistant spores in raw milk. High numbers of these spores were detected in the filter cloth of the milking equipment and in green crop and fodder samples. About 700 strains were isolated after the selective heating, of which 635 could be screened by fatty acid methyl ester analysis. Representative strains were subjected to amplified ribosomal DNA restriction analysis, 16S rRNA gene sequencing, percent G+C content, and DNA-DNA reassociations for further identification. The strain collection showed a remarkable diversity, with representatives of seven aerobic spore-forming genera. Bacillus licheniformis and Bacillus pallidus were the most predominant species overall. Twenty-three percent of the 603 spore-forming isolates proved to belong to 18 separate novel species. These findings suggest that the selective heating revealed a pool of unknown organisms with a higher heat-resistant character. This study showed that high spore counts can occur at the dairy farm and that feed and milking equipment can act as reservoirs or entry points for potentially highly heat-resistant spores into raw milk. Lowering this spore load by good hygienic measures could probably further reduce the contamination level of raw milk, in this way minimizing the aerobic spore-forming bacteria that could lead to spoilage of milk and dairy products. Assessment and characterization of this particular flora are of great importance to allow the dairy or food industry to adequately deal with newly arising microbiological problems.

Present address: Department of Bacteriology and Immunology, Veterinary and Agrochemical Research Centre, VAR-CODA-CERVA, 1180 Ukkel, Belgium.