The genera Actinobacillus, Haemophilus, and Pasteurella exhibit extensive polyphyletic branching in phylogenetic trees and do not represent coherent clusters of species. In this study, we have utilized molecular signatures identified through comparative genomic analyses in conjunction with genome based and multilocus sequence based phylogenetic analyses to clarify the phylogenetic and taxonomic boundary of these genera. We have identified large clusters of Actinobacillus, Haemophilus, and Pasteurella species which represent the "sensu stricto" members of these genera. We have identified 3, 7, and 6 conserved signature indels (CSIs), which are specifically shared by sensu stricto members of Actinobacillus, Haemophilus, and Pasteurella, respectively. We have also identified two different sets of CSIs that are unique characteristics of the pathogen containing genera Aggregatibacter and Mannheimia, respectively. It is now possible to demarcate the genera Actinobacillus sensu stricto, Haemophilus sensu stricto, and Pasteurella sensu stricto on the basis of discrete molecular signatures. The other members of the genera Actinobacillus, Haemophilus, and Pasteurella that do not fall within the "sensu stricto" clades and do not contain these molecular signatures should be reclassified as other genera. The CSIs identified here also provide useful diagnostic targets for the identification of current and novel members of the indicated genera.

Emergence and spread of antimicrobial resistance is a major concern for the dairy industry worldwide. Objectives were to determine: (1) phenotypic and genotypic prevalence of drug-specific resistance for 25 species of non-aureus staphylococci, and (2) associations between presence of resistance determinants and antimicrobial resistance. Broth micro-dilution was used to determine resistance profiles for 1,702 isolates from 89 dairy herds. Additionally, 405 isolates were sequenced to screen for resistance determinants. Antimicrobial resistance was clearly species-dependent. Resistance to quinupristin/dalfopristin was common in Staphylococcus gallinarum (prevalence of 98%), whereas S. cohnii and S. arlettae were frequently resistant to erythromycin (prevalence of 63 and 100%, respectively). Prevalence of resistance was 10% against β-lactams and tetracyclines. In contrast, resistance to antimicrobials critically important for human medicine, namely vancomycin, fluoroquinolones, linezolid and daptomycin, was uncommon (< 1%). Genes encoding multidrug-resistance efflux pumps and resistance-associated residues in deducted amino acid sequences of the folP gene were the most frequent mechanisms of resistance, regardless of species. The estimated prevalence of the mecA gene was 17% for S. epidermidis. Several genes, including blaZ, mecA, fexA, erm, mphC, msrA, and tet were associated with drug-specific resistance, whereas other elements were not. There were specific residues in gyrB for all isolates of species intrinsically resistant to novobiocin. This study provided consensus protein sequences of key elements previously associated with resistance for 25 species of non-aureus staphylococci from dairy cattle. These results will be important for evaluating effects of interventions in antimicrobial use in Canadian dairy herds.

Bacteriophages are bacterial parasites and are considered the most abundant and diverse biological entities on the planet. Previously we identified 154 prophages from 151 serovars of Salmonella enterica subsp. enterica. A detailed analysis of Salmonella prophage genomics is required given the influence of phages on their bacterial hosts and should provide a broader understanding of Salmonella biology and virulence and contribute to the practical applications of phages as vectors and antibacterial agents.

The rapid detection of foodborne microbial pathogens contaminating fresh fruits and vegetables during the intervening period between harvest and consumption could revolutionize microbial quality assurance of food usually consumed raw and those with a limited shelf life. We have developed a sensitive, shotgun whole genome sequencing protocol capable of detecting as few as 1 colony forming unit (cfu) of Salmonella enterica serovar Typhimurium spiked on 25 g of lettuce. The Ion Torrent sequencing platform was used to generate reads of globally amplified DNA from microbes recovered from the surface of lettuce followed by bioinformatic analyses of the nucleotide sequences to detect the presence of Salmonella. The test is rapid and sensitive, and appropriate for testing perishable foods, and those consumed raw, for Salmonella contamination. The test has the potential to be universally applicable to any microbial contaminant on lettuce as long as a suitable bioinformatics pipeline is available and validated. A universal test is expected to pave the way for preventive and precision food safety and the re-shaping of the entire spectrum of food safety investigations from the current disease-limiting, reactive procedure to a proactive, disease prevention process.

Non-aureus staphylococci (NAS) are the most frequently isolated pathogens from intramammary infection (IMI) in dairy cattle. Virulence factors (VFs) and mechanisms by which NAS cause IMI are not fully known. Herein, we analyzed the distribution of 191 VFs in 441 genomes of 25 NAS species, after classifying VFs into functional categories: adherence (n = 28), exoenzymes (n = 21), immune evasion (n = 20), iron metabolism (n = 29), and toxins (n = 93). In addition to establishing VF gene profiles, associations of VF genes between and among functional categories were computed, revealing distinctive patterns of association among VFs for various NAS species. Associations were also computed for low, medium, and high somatic cell count (SCC) and clinical mastitis (CM) isolates, demonstrating distinctive patterns of associations for low SCC and CM isolates, but no differences between high SCC and CM isolates. To determine whether VF distributions had any association with SCC or CM, various clustering approaches, including complete linkages, Ward clustering, and t-distributed stochastic neighbor embedding, were applied. However, no clustering of isolates representing low SCC, medium SCC, or high SCC or CM was identified. Regression analysis to test for associations with individual VF functional categories demonstrated that each additional toxin and host immune evasion gene increased the odds of having high SCC or CM, although an overall increase in the number of VFs was not associated with increased SCC or occurrence of CM. In conclusion, we established comprehensive VF gene profiling, determined VF gene distributions and associations, calculated pathogenic potentials of all NAS species, and detected no clear link between VF genes and mastitis. IMPORTANCE Non-aureus staphylococci (NAS) are the most frequently isolated pathogens from milk in dairy cattle worldwide. The virulence factors (VFs) and mechanisms by which these bacteria cause udder infection are not fully known. We determined the distribution and associations of 191 VFs in 25 NAS species and investigated the relationship between VFs and disease. Although the overall number of VFs was not associated with disease severity, increasing numbers of toxin and host immune evasion genes specifically were associated with more severe disease outcomes. These findings suggest that the development of disease and the interactions of VFs with the host are complex and determined by the interplay of genes rather than just the presence of virulence genes. Together, our results provide foundational genetic knowledge to other researchers to design and conduct further experiments, focusing on understanding the synergy between VFs and roles of individual NAS species in IMI and characterizing species-specific effects on udder health.

Digital dermatitis (DD) presents as painful, ulcerative or proliferative lesions that lead to bovine lameness affecting economic efficiency and animal welfare. Although DD etiological agent(s) have not been established, it is widely accepted that DD is a polymicrobial disease significantly associated with species of Treponema and the non-linear disease progression may be attributed to interactions among infecting bacteria. We postulated the morphological changes associated with DD lesion grades are related to interactions among infecting species of Treponema. We developed a novel species-specific qPCR that can identify the absolute abundance of the four of the most common species of Treponema in DD, T. phagedenis, T. medium, T. pedis and T. denticola, in a single reaction. We found species abundance and the number of distinct Treponema species present is higher in active, ulcerative lesions than in healing lesions, chronic lesions, and DD-free skin. Treponema spp. were present in both DD-free skin and M3 lesions following treatment with oxytetracycline. We have also found positive correlations among T. phagedenis, T. medium and T. pedis indicating they are significantly more likely to be found together than apart and their absolute quantities tend to increase together, a relationship which is not present with T. denticola. Further, we found Treponema, particularly viable T. denticola, in lesions 5 days post treatment with oxytetracycline (M3). Our findings suggest that pathogenicity may be closely associated with Treponema abundance, particularly T. phagedenis, T. medium and T. pedis, and interactions among them, independent of T. denticola. Our results provide a novel, consistent method to identify species of Treponema within DD lesions and associate Treponema spp. and abundance with morphological changes related to host pathogenicity.

Staphylococcus aureus, a common cause of bovine mastitis, is known for its ability to acquire to antimicrobial resistance and to secrete numerous virulence factors that can exacerbate inflammation. In addition, alpha-hemolysin has an important role in S. aureus infections, diversity of the hla gene (that produces alpha-hmolysin) in S. aureus isolated from bovine mastitis has not been well characterized. The objective was, therefore, to determine diversity of virulence genes, hla gene sequences, and clonal profiles of S. aureus from bovine mastitis in Chinese dairy herds, and to evaluate inter-relationships.

The complete genome sequences of 12 isolates of the rare Salmonella enterica serovar Adjame were determined by combining Nanopore and Illumina sequence reads. Chromosome sizes ranged from 4,597,011 bp to 4,678,052 bp, and the GC content was 52.3%. A virulent plasmid of 87,433 bp was found in only one isolate.

Staphylococcus aureus causes persistent clinical and subclinical bovine intramammary infections (IMI) worldwide. However, there is a lack of comprehensive information regarding genetic diversity, the presence of antimicrobial resistance (AMR), and virulence genes for S. aureus in bovine milk in Canada. Here, we performed whole-genome sequencing (WGS) of 119 Canadian bovine milk S. aureus isolates and determined they belonged to 8 sequence types (ST151, ST352, ST351, ST2187, ST2270, ST126, ST133, and ST8), 5 clonal complexes (CC151, CC97, CC126, CC133, and CC8), and 18 distinct Spa types. Pan-, core, and accessory genomes were composed of 6,340, 1,279, and 2,431 genes, respectively. Based on phenotypic screening for AMR, resistance was common against beta-lactams (19% of isolates) and sulfonamides (7% of isolates), whereas resistance against pirlimycin, tetracycline, ceftiofur, and erythromycin and to the combination of penicillin and novobiocin was uncommon (3, 3, 3, 2, and 2% of all isolates, respectively). We also determined distributions of 191 virulence factors (VFs) in 119 S. aureus isolates after classifying them into 5 functional categories (adherence [n = 28], exoenzymes [n = 21], immune evasion [n = 20], iron metabolism [n = 29], and toxins [n = 93]). Additionally, we calculated the pathogenic potential of distinct CCs and STs and determined that CC151 (ST151 and ST351) had the highest pathogenic potential (calculated by subtracting core-VFs from total VFs), followed by CC97 (ST352 and ST2187) and CC126 (ST126 and ST2270), potentially linked to their higher prevalence in bovine IMI worldwide. However, there was no statistically significant link between the presence of VF genes and mastitis.IMPORTANCE Staphylococcus aureus is a major cause of bovine intramammary infections, leading to significant economic losses to dairy industry in Canada and worldwide. There is a lack of knowledge regarding genetic diversity, the presence of antimicrobial resistance (AMR), and virulence genes for S. aureus isolated from bovine milk in Canada. Based on whole-genome sequencing and genomic analysis, we have determined the phylogeny and diversity of S. aureus in bovine milk and concluded that it had a large accessory genome, limited distribution of AMR genes, variable VF gene profiles and sequence types (ST), and clonal complex (CC)-specific pathogenic potentials. Comprehensive information on the population structure, as well as the virulence and resistance characteristics of S. aureus from bovine milk, will allow for source attribution, risk assessment, and improved therapeutic approaches in cattle.

Bovine digital dermatitis (DD) is a skin disorder that is a significant cause of infectious lameness in cattle around the world. However, very little is known about the etiopathogenesis of the disease and the microbiota associated with DD in beef cattle. In this study, we provide a comprehensive characterization of DD and healthy skin microbiota of feedlot beef cattle. We also developed and validated a novel multiplex quantitative PCR (qPCR) assay to quantify the distribution of DD-associated bacterial species across DD lesion stages. We determined the DD-associated microbiota with deep amplicon sequencing of the V3-V4 hypervariable region of the 16S rRNA gene, followed by the application of novel and existing qPCR assays to quantify species distributions of Treponema, Porphyromonas, Fusobacterium, and Bacteroides across lesion stages. Deep amplicon sequencing revealed that Treponema, Mycoplasma, Porphyromonas, and Fusobacterium were associated with DD lesions. Culturing of DD biopsy specimens identified Porphyromonas levii, Bacteroides pyogenes, and two Fusobacterium spp. within DD lesions. Using species-specific qPCR on DD lesion DNA, we identified P. levii in 100% of active lesion stages. Early-stage lesions were particularly associated with Treponema medium, T. phagedenis, and P. levii. This study suggests a core DD microbial group consisting of species of Treponema, Fusobacterium, Porphyromonas, and Bacteroides, which may be closely tied with the etiopathogenesis of DD. Further characterizations of these species and Mycoplasma spp. are necessary to understand the microbial factors involved in DD pathogenesis, which will help elucidate DD etiology and facilitate more targeted and effective mitigation and treatment strategies. IMPORTANCE Previous work, primarily in dairy cattle, has identified various taxa associated with digital dermatitis (DD) lesions. However, there is a significant gap in our knowledge of DD microbiology in beef cattle. In addition, characterization of bacteria at the species level in DD lesions is limited. In this study, we provide a framework for the accurate and reproducible quantification of major DD-associated bacterial species from DNA samples. Our findings support DD as a polymicrobial infection, and we identified a variety of bacterial species spanning multiple genera that are consistently associated with DD lesions. The DD-associated microbiota identified in this study may be capable of inducing the formation and progression of DD lesions and thus should be primary targets in future DD pathogenesis studies.

Non-aureus staphylococci (NAS), a heterogeneous group of a large number of species and subspecies, are the most frequently isolated pathogens from intramammary infections in dairy cattle. Phylogenetic relationships among bovine NAS species are controversial and have mostly been determined based on single-gene trees. Herein, we analyzed phylogeny of bovine NAS species using whole-genome sequencing (WGS) of 441 distinct isolates. In addition, evolutionary relationships among bovine NAS were estimated from multilocus data of 16S rRNA, hsp60, rpoB, sodA, and tuf genes and sequences from these and numerous other single genes/proteins. All phylogenies were created with FastTree, Maximum-Likelihood, Maximum-Parsimony, and Neighbor-Joining methods. Regardless of methodology, WGS-trees clearly separated bovine NAS species into five monophyletic coherent clades. Furthermore, there were consistent interspecies relationships within clades in all WGS phylogenetic reconstructions. Except for the Maximum-Parsimony tree, multilocus data analysis similarly produced five clades. There were large variations in determining clades and interspecies relationships in single gene/protein trees, under different methods of tree constructions, highlighting limitations of using single genes for determining bovine NAS phylogeny. However, based on WGS data, we established a robust phylogeny of bovine NAS species, unaffected by method or model of evolutionary reconstructions. Therefore, it is now possible to determine associations between phylogeny and many biological traits, such as virulence, antimicrobial resistance, environmental niche, geographical distribution, and host specificity.