title: Hypervariable pili and flagella genes provide suitable new targets for DNA high-resolution melt-based genotyping of dairy Geobacillus spp.
abstract: Although nonpathogenic in nature, spores of Geobacillus are able to attach to surfaces, germinate, and form biofilms, allowing rapid multiplication and persistence within milk powder processing plants, causing final product contamination, and eventually leading to a loss of revenue in terms of downgraded product quality. As a result, Geobacillus spp. have been found to be common contaminants of milk powder worldwide. Genotyping methods can help in gaining insight into the ecology and transmission of these thermophilic bacteria within and between dairy processing plants. The objective of this study was to use the assembled draft genomes of two Geobacillus spp. to identify and test new hypervariable genotyping targets for differentiating closely related dairy Geobacillus isolates. The two Geobacillus spp. strains obtained from high spore count powders were obtained in 2010 (isolate 7E) and in 1995 (isolate 126) and were previously shown to be of same genotype based on a variable number tandem repeat genotyping method. Significant nucleotide sequence variation was found in genes encoding pili and flagella, which were further investigated as suitable loci for a new high-resolution melt analysis (HRMA)-based genotyping method. Three genes encoding pulG (containing prepilin-type N-terminal cleavage domain), pilT (pili retraction protein), and fliW (flagellar assembly protein) were selected as targets for the new pili/flagella gene (PilFla) HRMA genotyping method. The three-gene-based PilFla-HRMA genotyping method differentiated 35 milk powder Geobacillus spp. isolates into 19 different genotype groups (D = 0.93), which compared favorably to the previous method (which used four variable number tandem repeat loci) that generated 16 different genotype groups (D = 0.90). In conclusion, through comparative genomics of two closely related dairy Geobacillus strains, we have identified new hypervariable regions that prove to be useful targets for highly discriminatory genotyping.
authors.affiliation: School of Agriculture and Food Sciences, The University of Queensland, St. Lucia Campus, Brisbane, Queensland 4072, Australia.
authors.forename: Kanika
authors.initials: K
authors.lastname: Chauhan
authors.affiliation: School of Agriculture and Food Sciences, Auckland University of Technology, City Campus, Auckland 1010, New Zealand.
authors.forename: R Brent
authors.initials: RB
authors.lastname: Seale
authors.affiliation: School of Agriculture and Food Sciences, The University of Queensland, St. Lucia Campus, Brisbane, Queensland 4072, Australia.
authors.forename: Hilton C
authors.initials: HC
authors.lastname: Deeth
authors.affiliation: School of Agriculture and Food Sciences, Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, St. Lucia Campus, Brisbane, Queensland 4072, Australia. m.turner2@uq.edu.au.
authors.forename: Mark S
authors.initials: MS
authors.lastname: Turner
mesh_terms: D000818:Animals; D001483:Base Sequence; D004269:DNA, Bacterial / Q000235:genetics; D003611:Dairy Products / Q000382:microbiology; D010861:Fimbriae, Bacterial / Q000235:genetics*; D005407:Flagella / Q000235:genetics*; D005516:Food Microbiology / Q000379:methods*; D016680:Genome, Bacterial; D060005:Genotyping Techniques; D056506:Geobacillus / Q000145:classification* / Q000235:genetics; D008892:Milk / Q000382:microbiology; D018598:Minisatellite Repeats; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012689:Sequence Homology, Nucleic Acid; D013045:Species Specificity
chemical_list: D004269:DNA, Bacterial