Psychrotolerant spore-forming bacteria represent a major challenge to the goal of extending the shelf life of pasteurized dairy products. The objective of this study was to identify prominent phylogenetic groups of dairy-associated aerobic sporeformers and to characterize representative isolates for phenotypes relevant to growth in milk. Analysis of sequence data for a 632-nucleotide fragment of rpoB showed that 1,288 dairy-associated isolates (obtained from raw and pasteurized milk and from dairy farm environments) clustered into two major divisions representing (i) the genus Paenibacillus (737 isolates, including the species Paenibacillus odorifer, Paenibacillus graminis, and Paenibacillus amylolyticus sensu lato) and (ii) Bacillus (n ‫؍‬ 467) (e.g., Bacillus licheniformis sensu lato, Bacillus pumilus, Bacillus weihenstephanensis) and genera formerly classified as Bacillus (n ‫؍‬ 84) (e.g., Viridibacillus spp.). When isolates representing the most common rpoB allelic types (ATs) were tested for growth in skim milk broth at 6°C, 6/9 Paenibacillus isolates, but only 2/8 isolates representing Bacillus subtypes, grew >5 log CFU/ml over 21 days. In addition, 38/40 Paenibacillus isolates but only 3/47 Bacillus isolates tested were positive for ␤-galactosidase activity (including some isolates representing Bacillus licheniformis sensu lato, a common dairy-associated clade). Our study confirms that Paenibacillus spp. are the predominant psychrotolerant sporeformers in fluid milk and provides 16S rRNA gene and rpoB subtype data and phenotypic characteristics facilitating the identification of aerobic spore-forming spoilage organisms of concern. These data will be critical for the development of detection methods and control strategies that will reduce the introduction of psychrotolerant sporeformers and extend the shelf life of dairy products.

Streptococcus bovis HC5 cultures released a broad spectrum lantibiotic, bovicin HC5, into the cell-fee culture supernatant after they reached stationary phase, but most of the antibacterial activity remained cell-associated. Cell-associated bovicin HC5 was more resistant to degradation by Pronase E than the cell-free activity. Acidic NaCl (pH 2.0, 100 mM) did not release all of the cell-associated activity, and cells that were sequentially treated with acidic NaCl and Pronase E still had antibacterial activity. Cell-associated activity retained after acidic NaCl treatment was still able to catalyze potassium efflux from S. bovis JB1, a sensitive strain. These results indicate that cell-associated bovicin HC5 is more active and stable than cell-free bovicin HC5.

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A bacteriocin-producing Streptococcus bovis strain (HC5) outcompeted a sensitive strain (JB1) before it reached stationary phase (pH 6.4), even though it grew 10% slower and cell-free bovicin HC5 could not yet be detected. The success of bacteriocin-negative S. bovis isolates was enhanced by the presence of another sensitive bacterium (Clostridium sticklandii SR). PCR based on repetitive DNA sequences indicated that S. bovis HC5 was not simply transferring bacteriocin genes to S. bovis JB1. When the two S. bovis strains were coinoculated into minimal medium, bacteriocin-negative isolates predominated, and this effect could be explained by the longer lag time (0.5 vs. 1.5 h) of S. bovis HC5. If the glucose concentration of the minimal medium was increased from 2 to 7 mg mL(-1), the effect of lag time was diminished and bacteriocin-producing isolates once again dominated the coculture. When the competition was examined in continuous culture, it became apparent that batch culture inocula were never able to displace a strain that had already reached steady state, even if the inoculum was large. This result indicated that bacterial selection for substrate affinity was even more important than bacteriocin production.