Paenibacillus odorifer, the Predominant Paenibacillus Species Isolated from Milk in the United States, Demonstrates Genetic and Phenotypic Conservation of Psychrotolerance but Clade-Associated Differences in Nitrogen Metabolic Pathways

Although Paenibacillus species isolates are frequently isolated from pasteurized fluid milk, the link between the genetic diversity and phenotypic characteristics of these isolates was not well understood, especially as some Bacillales isolated from milk are unable to grow at refrigeration temperatures. Our data demonstrate that Paenibacillus spp. isolated from fluid milk represent tremendous interspecies diversity, with P. odorifer being the predominant Paenibacillus sp. isolated. Furthermore, genetic and phenotypic data support that P. odorifer is well suited to transition from a soil-dwelling environment, where nitrogen fixation (and other nitrate/nitrite reduction pathways present only in clade A) may facilitate growth, to fluid milk, where its multiple cold shock-associated adaptations enable it to grow at refrigeration temperatures throughout the storage of milk. Therefore, efforts to reduce bacterial contamination of milk will require a systematic approach to reduce P. odorifer contamination of raw milk. ABSTRACT Paenibacillus is a spore-forming bacterial genus that is frequently isolated from fluid milk and is proposed to play a role in spoilage. To characterize the genetic and phenotypic diversity of Paenibacillus spp. , we first used rpoB allelic typing data for a preexisting collection of 1,228 Paenibacillus species isolates collected from raw and processed milk, milk products, and dairy environmental sources. Whole-genome sequencing (WGS) and average nucleotide identity by BLAST (ANIb) analyses performed for a subset of 58 isolates representing unique and overrepresented rpoB allelic types in the collection revealed that these isolates represent 21 different Paenibacillus spp. , with P. odorifer being the predominant species. Further genomic characterization of P. odorifer isolates identified two distinct phylogenetic clades, clades A and B, which showed significant overrepresentation of 172 and 164 ortholog clusters and 94 and 52 gene ontology (GO) terms, respectively. While nitrogen fixation genes were found in both clades, multiple genes associated with nitrate and nitrite reduction were overrepresented in clade A isolates; additional phenotypic testing demonstrated that nitrate reduction is specific to isolates in clade A. Hidden Markov models detected 9 to 10 different classes of cold shock-associated genetic elements in all P. odorifer isolates. Phenotypic testing revealed that all isolates tested here can grow in skim milk broth at 6°C, suggesting that psychrotolerance is conserved in P. odorifer. Overall, our data suggest that Paenibacillus spp. isolated from milk in the United States represent broad genetic diversity, which may provide challenges for targeted-control strategies aimed at reducing fluid milk spoilage. IMPORTANCE Although Paenibacillus species isolates are frequently isolated from pasteurized fluid milk, the link between the genetic diversity and phenotypic characteristics of these isolates was not well understood, especially as some Bacillales isolated from milk are unable to grow at refrigeration temperatures. Our data demonstrate that Paenibacillus spp. isolated from fluid milk represent tremendous interspecies diversity, with P. odorifer being the predominant Paenibacillus sp. isolated. Furthermore, genetic and phenotypic data support that P. odorifer is well suited to transition from a soil-dwelling environment, where nitrogen fixation (and other nitrate/nitrite reduction pathways present only in clade A) may facilitate growth, to fluid milk, where its multiple cold shock-associated adaptations enable it to grow at refrigeration temperatures throughout the storage of milk. Therefore, efforts to reduce bacterial contamination of milk will require a systematic approach to reduce P. odorifer contamination of raw milk.