Short communication: Typing and tracking Bacillaceae in raw milk and milk powder using pyroprinting.

Contamination of fluid and processed milk products with endospore-forming bacteria, such as Bacillaceae, affect milk quality and longevity. Contaminants come from a variety of sources, including the dairy farm environment, transportation equipment, or milk processing machinery. Tracking the origin of bacterial contamination to allow specifically targeted remediation efforts depends on a reliable strain-typing method that is reproducible, fast, easy to use, and amenable to computerized analysis. Our objective was to adapt a recently developed genotype-based Escherichia coli strain-typing method, called pyroprinting, for use in a microbial source-tracking study to follow endospore-forming bacillus bacteria from raw milk to powdered milk. A collection of endospores was isolated from both raw milk and its finished powder, and, after germination, the vegetative cells were subject to the pyroprinting protocol. Briefly, a ribosomal DNA intergenic transcribed spacer present in multiple copies in Bacillaceae genomes was amplified by the PCR. This multicopy locus generated a mixed PCR product that was subsequently subject to pyrosequencing, a quantitative real-time sequencing method. Through a series of enzymatic reactions, each nucleotide incorporation event produces a photon of light that is quantified at each nucleotide dispensation. The pattern of light peaks generated from this mixed template reaction is called a pyroprint. Isolates with pyroprints that match with a Pearson correlation of 0.99 or greater are considered to be in the same group. The pyroprint also contains some sequence data useful for presumptive species-level identification. This method identified groups with isolates from raw milk only, from powdered milk only, or from both sources. This study confirms pyroprinting as a rapid, reproducible, automatically digitized tool that can be used to distinguish bacterial strains into taxonomically relevant groups and, thus, indicate probable origins of bacterial contamination in powdered milk.