title: Short communication: Typing and tracking Bacillaceae in raw milk and milk powder using pyroprinting.
abstract: 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.
authors.affiliation: Center for Applications in Biotechnology, California Polytechnic State University, San Luis Obispo 93407.
authors.forename: Jennifer J
authors.initials: JJ
authors.lastname: VanderKelen
authors.affiliation: Synthetic Genomics Inc., La Jolla, CA 92037.
authors.forename: Ryan D
authors.initials: RD
authors.lastname: Mitchell
authors.affiliation: Department of Chemistry and Biochemistry, California Polytechnic State University, San Luis Obispo 93407.
authors.forename: Andrea
authors.initials: A
authors.lastname: Laubscher
authors.affiliation: Center for Applications in Biotechnology, California Polytechnic State University, San Luis Obispo 93407; Department of Biological Sciences, California Polytechnic State University, San Luis Obispo 93407.
authors.forename: Michael W
authors.initials: MW
authors.lastname: Black
authors.affiliation: Center for Applications in Biotechnology, California Polytechnic State University, San Luis Obispo 93407; Department of Chemistry and Biochemistry, California Polytechnic State University, San Luis Obispo 93407.
authors.forename: Anya L
authors.initials: AL
authors.lastname: Goodman
authors.affiliation: Workday, Pleasanton, CA 94588.
authors.forename: Aldrin K
authors.initials: AK
authors.lastname: Montana
authors.affiliation: Center for Applications in Biotechnology, California Polytechnic State University, San Luis Obispo 93407; Department of Computer Science, California Polytechnic State University, San Luis Obispo 93407.
authors.forename: Alexander M
authors.initials: AM
authors.lastname: Dekhtyar
authors.affiliation: Center for Applications in Biotechnology, California Polytechnic State University, San Luis Obispo 93407; Department of Dairy Science, California Polytechnic State University, San Luis Obispo 93407.
authors.forename: Rafael
authors.initials: R
authors.lastname: Jimenez-Flores
authors.affiliation: Center for Applications in Biotechnology, California Polytechnic State University, San Luis Obispo 93407; Department of Biological Sciences, California Polytechnic State University, San Luis Obispo 93407. Electronic address: ckitts@calpoly.edu.
authors.forename: Christopher L
authors.initials: CL
authors.lastname: Kitts
mesh_terms: D000818:Animals; D001406:Bacillaceae / Q000145:classification* / Q000302:isolation & purification*; D015373:Bacterial Typing Techniques; D004269:DNA, Bacterial / Q000235:genetics; D005511:Food Handling; D008892:Milk / Q000382:microbiology*; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D013171:Spores, Bacterial / Q000302:isolation & purification
chemical_list: D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S
keywords: Bacillaceae; biotechnology; microbial source tracking; strain typing