Phylogenetic analysis and characterization of bacterial sporeformer isolates obtained from raw milk, pasteurized milk, and dairy farm environments R. A. Ivy, M. L. Ranieri, N. H. Martin, H. den Bakker, B. M. Xavier, M. Wiedmann, and K. J. Boor Milk Quality Improvement Program 200 Stocking Hall Ithaca, NY ph: f: Figure 1. Maximum likelihood phylogenetic tree of rpoB allelic types (AT) of Bacillus spp. (A) and Paenibacillus spp. (B) isolated from pasteurized milk (red wedge) raw milk (blue wedge) and dairy farm environments (green wedge). Numerical node labels values represent the percentage of bootstrap replications that supported the respective node. Only bootstrap values greater than 60 are shown. Species IDs are based on 16S sequencing. The sensu lato (s.l.) notation is used for clades that contained isolates matching (i.e. > 98% sequence similarity) more than one type strain 16S sequence. The confer (cf.) designation is used to denote isolates that are similar (97-98% sequence similarity) to more than one 16S type sequence. Abstract* The presence of psychrotolerant endospore-forming bacteria represents a major challenge to extending the shelf life of pasteurized dairy products. The objective of this study was to identify prominent phylogenetic groups of dairy-associated aerobic sporeformers (i.e., isolates from raw and pasteurized milk, and dairy farm environments) and characterize representative isolates for phenotypes relevant to cold growth in milk. All isolates (n = 1288) were classified within the family Bacillaceae. Frequently isolated clades consisted of Bacillus spp. (n = 467; e.g., B. licheniformis s.l., B. pumilis, and B. weihenstephanensis), genera formerly classified as Bacillus (n = 84; e.g., Viridibacillus spp.) and Paenibacillus spp. (n = 737; e.g., P. odorifer, P. graminis, and P. amylolyticus). Only two out of nine isolates representing prominent non-Paenibacillus subtypes [as determined by rpoB allelic typing (AT)] grew to > 4 log(CFU/ml) in skim milk broth (SMB) at 6  C, whereas all but two out of Paenibacillus representative isolates grew to > 4 log(CFU/ml) in SMB at 6  C. Though most Paenibacillus isolates were positive for β-galactosidase activity at 32°C and most non-Paenibacillus were negative, isolates representative of Bacillus licheniformis s.l. AT1 (13% of non-Paenibacillus isolates) showed varying β- galactosidase activity. Therefore β-galactosidase activity alone cannot reliably distinguish Paenibacillus from other Bacillaceae. Our study confirmed that Paenibacillus spp. are the predominant psychrotolerant sporeformers in fluid milk and provided molecular subtype organization and phenotypic characteristics of prominent clades of aerobic sporeformers. This study, thus, contributes to the understanding of fluid milk bacterial ecology and will facilitate the development of sporeformer prevention methods aimed at extending the shelf life of pasteurized dairy foods. Introduction Materials and Methods Results and Discussion Conclusions References According to the International Dairy Foods Association, the U.S. per annum fluid milk purchases total 6 billion gallons (6). As much as 20% of this is discarded prior to consumption, due in part to microbial spoilage (7). Psychrotolerant, or “cold-thriving”, Gram-positive sporeformers have the potential to survive conventional pasteurization and can grow during refrigerated storage, resulting in off flavors and curdling in the final product. Bacillus and Paenibacillus have been identified as the prominent genera of Gram-positive sporeformers in pasteurized fluid milk (9). While both Bacillus spp. and Paenibacillus spp. have been traced from dairy farm environments, through processing systems, to pasteurized milk (9), Bacillus spp. are predominantly detected early during the shelf-life of pasteurized milk, whereas Paenibacillus has been shown to predominate late in shelf-life (9). Therefore, though both species are present directly after processing, Paenibacillus spp. are likely to predominate in refrigerated pasteurized fluid milk. Currently, due to a lack of information on the population structure and genotypes of dairy-associated sporeformers, no methods exist to differentiate Bacillus from Paenibacillus. Figure 2. Growth of isolates representing prominent rpoB allelic types of non-Paenibacillus Bacillaceae (a) and Paenibacillus (b) in skim milk broth at 6  C. Bacillus clades tested were B. licheniformis s.l. 1 (AT001), B. weihenstephanensis (AT003), Viridibacillus spp. (AT17), B. pumilis 1(AT20), B. aerophilus s.l. (AT135), B. safensis (AT141), and B. cereus s.l. 1 (AT158). Paenibacillus clades tested were P. odorifer 1 (AT15), P. amylolyticus s.l. (AT23 and AT111), P. graminis 2 (AT39), P. graminis 1 (AT45), P. xylanilyticus s.l. (AT100), P. cf. peoriae (AT157), P. lautus (AT159), and P. odorifer 3 (AT260). A - Bacillus B - Paenibacillus A - Bacillus Isolate collection and selection. Gram-positive sporeformer isolates were collected from several studies, which employed standard methods for the examination of dairy products, (3-5, 8, 10). Spore treatments (80  C for 12 min) were conducted to eliminate vegetative cells. Colonies representing each visually distinct morphology were streaked for isolation on BHI agar total isolates were cataloged. rpoB sequencing. Molecular subtyping of all isolates was performed based on the DNA sequence for a 632-nucleotide (nt) fragment (nt 2455 to 3086) of the rpoB gene. Briefly, the rpoB fragment was amplified using PCR primers that were previously described (1) and PCR conditions detailed by Durak et al. (2). rpoB PCR products were purified using the QIAquick PCR Purification Kit and sequencing was performed at Cornell University’s Life Sciences Core Laboratory Center (Ithaca, N.Y.). AT assignment. A unique rpoB allelic type (AT) was assigned to a gene sequence that differed from any previously obtained sequence by one or more nucleotides. Isolates with different ATs were considered to represent different subtypes. 283 unique rpoB ATs were identified. Alignment, tree construction, and species identification. rpoB sequences were trimmed and aligned. An rpoB maximum likelihood (ML) phylogenetic tree was constructed using the rapid maximum likelihood algorithm RAxML with rapid bootstrapping (100 bootstrap replicates). For species identification, partial 16S rDNA sequences obtained for each unique rpoB allelic type were compared against type-strain 16S rDNA sequences using the “Seqmatch” function the Ribosomal Database Project (RDP) database ( Cold Growth. A single colony was inoculated into 5 ml of BHI broth. Serial dilutions of the culture were performed and 1 mL was transferred into 9 mL of sterile Skim Milk Broth (SMB) for a final inoculum level of ~10 2 CFU/ml. SMB samples were plated immediately and after 6, 10, 13 17, 20, and 24 days of incubation at 6°C.  -galactosidase (  -gal) activity. Cultures were streaked onto BHI agar with and without an overlay of 100  l of a solution of bromo-chloro-indolyl-galactopyranoside (X-gal; 40  g/ml) and incubated at 32  C for 24 h. Blue colonies on the plates containing X-gal were indicative that the isolate was positive for  -galactosidase activity. Prominent dairy-associated Bacillus clades. B. pumilis (Group I) B. licheniformis s.l. (Group II), B. cereus s.l., or B. weihenstephanensis (Both in Group III), together, represented 337 out of 685 (49%) of Bacillus isolates in our study (Fig 1a). Group IV consists of isolates identified as belonging to genera formerly classified as Bacillus (i.e., Bacillus sensu lato), including Viridibacillus spp., which accounted for 46 isolates (Fig 1a). Most Paenibacillus isolates were positive for β-gal activity, whereas most Bacillus clades were not. β-gal is required for the metabolism of the milk carbohydrate lactose. In general, representative isolates of Bacillus spp. were negative for β-gal activity, whereas representative Paenibacillus isolates were positive (Table 1). However, representatives of the most frequently isolated Bacillus species (B. licheniformis) showed varying activity (Table1). Representatives from prominent Paenibacillus clades grow in milk during refrigeration, whereas, with the exception of B. weihenstephanensis, representatives from prominent Bacillus clades do not. Representative isolates of the B. weihenstephanensis [allelic type (AT) 3], and Viridibacillus (AT17) were the only non-Paenibacillus isolates to reach > 4.0 log CFU/ml by 21 d (Fig 2a), while all but two representative Paenibacillus isolates (AT17 and AT100) reached 4.0 log CFU/ml by 21 d (Fig 2b). Though both Bacillus spp. and Paenibacillus spp. are commonly isolated from dairy systems, a small number of Paenibacillus species account for the majority of psychrotolerant sporeformers in pasteurized milk β-galactosidase activity can be used as a preliminary screen for Paenibacillus, but cannot reliably discriminate Paenibacillus from Bacillus. Identification of Paenibacillus-specific targets will facilitate the development of technologies to prevent the introduction of psychrotolerant sporeformers into pasteurized milk Prominent dairy associated Paenibacillus clades. Within Paenibacillus isolates (n = 737), six major clades, each consisting of a single species ID accounted for 677 (92%) of Paenibacillus isolates. These clades are P. odorifer 1-3, P. graminis, P. cf. peoriae and P. amylolyticus s.l. Therefore, a relatively small number of species and clades represent the majority of dairy- associated aerobic sporeformers. B - Paenibacillus 1. Drancourt, M., V. Roux, P. E. Fournier, and D. Raoult J Clin Microbiol 42: Durak, Z., H. Fromm, J. Huck, R. Zadoks, and K. Boor J Food Sci 71:M50-M Huck, J. R., B. H. Hammond, S. C. Murphy, N. H. Woodcock, and K. J. Boor J Dairy Sci 90: Huck, J. R., M. Sonnen, and K. J. Boor J Dairy Sci 91: Huck, J. R., N. H. Woodcock, R. D. Ralyea, and K. J. Boor J Food Prot 70: International Dairy Foods Association Dairy Facts: 2010 Edition. 7. Kantor, L. C., K. LIpton, A. Manchester, and V. Oliveira Food Review 20: Ranieri, M. L., and K. J. Boor J Dairy Sci 92: Ranieri, M. L., and K. J. Boor Aust J Dairy Technol 65: Ranieri, M. L., J. R. Huck, M. Sonnen, D. M. Barbano, and K. J. Boor J Dairy Sci 92: Acknowledgements The contributions of the staff of the Milk Quality Improvement Program (MQIP) at Cornell University are acknowledged. This work is supported by the New York State Milk Promotion Advisory Board through the New York State Department of Agriculture and New York State dairy farmers Objectives The objective of this study was to identify prominent dairy-associated psychrotolerant sporeformers by: 1.Using rpoB sequencing to systematically identify prominent clades of dairy-associated sporeformers 2.Determining relevant phenotypic characteristics (i.e., growth in milk during refrigerated storage and  - galactosidase activity) for representatives of prominent clades Table 1. Frequency of isolation and β-galactosidase activity of rpoB clades isolated more than ten times Clade ID# IsolatesRepresentative ATFSL ID a β-gal activity b Bacillus aerophilus s.l.24135H Bacillus licheniformis s.l F4-073+/- Bacillus pumilis 15272H Bacillus safensis30141H Bacillus subtilis1765H Bacillus weihenstephanensis503F Viridibacillus spp.4617F Paenibacillus amylolyticus s.l.9623F Paenibacillus cf. peoriae24157H8-551wp Paenibacillus graminis 12345H Paenibacillus graminis 22339H Paenibacillus odorifer F Paenibacillus odorifer R Paenibacillus c.f. xylanilyticus11100H a Cornell Food Safety Lab isolate identifiation. Additional isolate information available at b Indicates whether every representative isolate tested from this group was positive (+) or negative (-) for β-gal activity or whether the group had both positive and negative representatives (+/-) or had at least one isolate that showed weakly positive (wp) activity