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Background: Sporeformers in the order Bacillales are important contributors to spoilage of pasteurized milk. While only a few Bacillus and Viridibacillus strains can grow in milk at 6°C, the majority of Paenibacillus isolated from pasteurized fluid milk can grow under these conditions. To gain a better understanding of genomic features of these important spoilage organisms and to identify candidate genomic features that may facilitate cold growth in milk, we performed a comparative genomic analysis of selected dairy associated sporeformers representing isolates that can and cannot grow in milk at 6°C., Results: The genomes for seven Paenibacillus spp., two Bacillus spp., and one Viridibacillus sp. isolates were sequenced. Across the genomes sequenced, we identified numerous genes encoding antimicrobial resistance mechanisms, bacteriocins, and pathways for synthesis of non-ribosomal peptide antibiotics. Phylogenetic analysis placed genomes representing Bacillus, Paenibacillus and Viridibacillus into three distinct well supported clades and further classified the Paenibacillus strains characterized here into three distinct clades, including (i) clade I, which contains one strain able to grow at 6°C in skim milk broth and one strain not able to grow under these conditions, (ii) clade II, which contains three strains able to grow at 6°C in skim milk broth, and (iii) clade III, which contains two strains unable to grow under these conditions. While all Paenibacillus genomes were found to include multiple copies of genes encoding β-galactosidases, clade II strains showed significantly higher numbers of genes encoding these enzymes as compared to clade III strains. Genome comparison of strains able to grow at 6°C and strains unable to grow at this temperature identified numerous genes encoding features that might facilitate the growth of Paenibacillus in milk at 6°C, including peptidases with cold-adapted features (flexibility and disorder regions in the protein structure) and cold-adaptation related proteins (DEAD-box helicases, chaperone DnaJ)., Conclusions: Through a comparative genomics approach we identified a number of genomic features that may relate to the ability of selected Paenibacillus strains to cause spoilage of refrigerated fluid milk. With additional experimental evidence, these data will facilitate identification of targets to detect and control Gram positive spore formers in fluid milk.

Background: Salmonella is a widely distributed foodborne pathogen that causes tens of millions of salmonellosis cases globally every year. While the genomic diversity of Salmonella is increasingly well studied, our knowledge of Salmonella phage genomic diversity is still rather limited, despite the contributions of both lysogenic and lytic phages to Salmonella virulence, diversity and ecology (e.g., through horizontal gene transfer and Salmonella lysis). To gain a better understanding of phage diversity in a specific ecological niche, we sequenced 22 Salmonella phages isolated from a number of dairy farms from New York State (United States) and analyzed them using a comparative genomics approach., Results: Classification of the 22 phages according to the presence/absence of orthologous genes allowed for classification into 8 well supported clusters. In addition to two phage clusters that represent novel virulent Salmonella phages, we also identified four phage clusters that each contained previously characterized phages from multiple continents. Our analyses also identified two clusters of phages that carry putative virulence (e.g., adhesins) and antimicrobial resistance (tellurite and bicyclomycin) genes as well as virulent and temperate transducing phages. Insights into phage evolution from our analyses include (i) identification of DNA metabolism genes that may facilitate nucleotide synthesis in phages with a G+C % distinct from Salmonella, and (ii) evidence of Salmonella phage tailspike and fiber diversity due to both single nucleotide polymorphisms and major re-arrangements, which may affect the host specificity of Salmonella phages., Conclusions: Genomics-based characterization of 22 Salmonella phages isolated from dairy farms allowed for identification of a number of novel Salmonella phages. While the comparative genomics analyses of these phages provide a number of new insights in the evolution and diversity of Salmonella phages, they only represent a first glimpse into the diversity of Salmonella phages that is likely to be discovered when phages from different environments are characterized.

As the development of molecular serotyping approaches is critical for Salmonella spp., which include >2,600 serovars, we performed an initial evaluation of the ability to identify Salmonella serovars using (i) different molecular subtyping methods and (ii) a newly implemented combined PCR- and sequencing-based approach that directly targets O- and H-antigen-encoding genes. Initial testing was performed using 46 isolates that represent the top 40 Salmonella serovars isolated from human and nonhuman sources, as reported by the U.S. Centers for Disease Control and Prevention and the World Health Organization. Multilocus sequence typing (MLST) was able to accurately predict the serovars for 42/46 isolates and showed the best ability to predict serovars among the subtyping methods tested. Pulsed-field gel electrophoresis (PFGE), ribotyping, and repetitive extragenic palindromic sequence-based PCR (rep-PCR) were able to accurately predict the serovars for 35/46, 34/46, and 30/46 isolates, respectively. Among the methods, S. enterica subsp. enterica serovars 4,5,12:i:-, Typhimurium, and Typhimurium var. 5- were frequently not classified correctly, which is consistent with their close phylogenetic relationship. To develop a PCR- and sequence-based serotyping approach, we integrated available data sources to implement a combination PCR-based O-antigen screening and sequencing of internal fliC and fljB fragments. This approach correctly identified the serovars for 42/46 isolates in the initial set representing the most common Salmonella serovars, as well as for 54/63 isolates representing less common Salmonella serovars. Our study not only indicates that different molecular approaches show the potential to allow for rapid serovar classification of Salmonella isolates, but it also provides data that can help with the selection of molecular serotyping methods to be used by different laboratories.

The genetic diversity represented by >2,500 different Salmonella serovars provides a yet largely uncharacterized reservoir of mobile elements that can contribute to the frequent emergence of new pathogenic strains of this important zoonotic pathogen. Currently, our understanding of Salmonella mobile elements is skewed by the fact that most studies have focused on highly virulent or common serovars. To gain a more global picture of mobile elements in Salmonella, we used prediction algorithms to screen for mobile elements in 16 sequenced Salmonella genomes representing serovars for which no prior genome scale mobile element data were available. From these results, selected mobile elements underwent further analyses in the form of validation studies, comparative analyses, and PCR-based population screens. Through this analysis we identified a novel plasmid that has two cointegrated replicons (IncI1-IncFIB); this plasmid type was found in four genomes representing different Salmonella serovars and contained a virulence gene array that had not been previously identified. A Salmonella Montevideo isolate contained an IncHI and an IncN2 plasmid, which both encoded antimicrobial resistance genes. We also identified two novel genomic islands (SGI2 and SGI3), and 42 prophages with mosaic architecture, seven of them harboring known virulence genes. Finally, we identified a novel integrative conjugative element (ICE) encoding a type IVb pilus operon in three non-typhoidal Salmonella serovars. Our analyses not only identified a considerable number of mobile elements that have not been previously reported in Salmonella, but also found evidence that these elements facilitate transfer of genes that were previously thought to be limited in their distribution among Salmonella serovars. The abundance of mobile elements encoding pathogenic properties may facilitate the emergence of strains with novel combinations of pathogenic traits.

Background: Divergence of bacterial populations into distinct subpopulations is often the result of ecological isolation. While some studies have suggested the existence of Salmonella enterica subsp. enterica subclades, evidence for these subdivisions has been ambiguous. Here we used a comparative genomics approach to define the population structure of Salmonella enterica subsp. enterica, and identify clade-specific genes that may be the result of ecological specialization., Results: Multi-locus sequence analysis (MLSA) and single nucleotide polymorphisms (SNPs) data for 16 newly sequenced and 30 publicly available genomes showed an unambiguous subdivision of S. enterica subsp. enterica into at least two subpopulations, which we refer to as clade A and clade B. Clade B strains contain several clade-specific genes or operons, including a β-glucuronidase operon, a S-fimbrial operon, and cell surface related genes, which strongly suggests niche specialization of this subpopulation. An additional set of 123 isolates was assigned to clades A and B by using qPCR assays targeting subpopulation-specific SNPs and genes of interest. Among 98 serovars examined, approximately 20% belonged to clade B. All clade B isolates contained two pathogenicity related genomic islands, SPI-18 and a cytolethal distending toxin islet; a combination of these two islands was previously thought to be exclusive to serovars Typhi and Paratyphi A. Presence of β-glucuronidase in clade B isolates specifically suggests an adaptation of this clade to the vertebrate gastrointestinal environment., Conclusions: S. enterica subsp. enterica consists of at least two subpopulations that differ specifically in genes involved in host and tissue tropism, utilization of host specific carbon and nitrogen sources and are therefore likely to differ in ecology and transmission characteristics.

Non-typhoidal Salmonella represents an important human and animal pathogen world-wide. Most human salmonellosis cases are foodborne, but each year infections are also acquired through direct or indirect animal contact in homes, veterinary clinics, zoological gardens, farm environments or other public, professional or private settings. Clinically affected animals may exhibit a higher prevalence of shedding than apparently healthy animals, but both can shed Salmonella over long periods of time. In addition, environmental contamination and indirect transmission through contaminated food and water may complicate control efforts. The public health risk varies by animal species, age group, husbandry practice and health status, and certain human subpopulations are at a heightened risk of infection due to biological or behavioral risk factors. Some serotypes such as Salmonella Dublin are adapted to individual host species, while others, for instance Salmonella Typhimurium, readily infect a broad range of host species, but the potential implications for human health are currently unclear. Basic hygiene practices and the implementation of scientifically based management strategies can efficiently mitigate the risks associated with animal contacts. However, the general public is frequently unaware of the specific disease risks involved, and high-risk behaviors are common. Here we describe the epidemiology and serotype distribution of Salmonella in a variety of host species. In addition, we review our current understanding of the public health risks associated with different types of contacts between humans and animals in public, professional or private settings, and, where appropriate, discuss potential risk mitigation strategies.