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This announcement reports the complete genome sequence of a non-Shiga toxin-producing Escherichia coli strain that was isolated from municipal biosolids collected from a Canadian wastewater treatment plant. This strain contains multiple metal, antimicrobial, and heat resistance genes, as determined by genome sequencing, and could be a useful bacterial model for future studies.

Staphylococcus aureus is one of the most important bacterial pathogens causing bovine mastitis, which leads to huge economic losses worldwide. Here, we report draft genome sequences and antimicrobial resistance gene profiles of five Staphylococcus aureus strains that were isolated from bovine milk in Pakistan.

Listeria monocytogenes , a Gram-positive, rod-shaped, non-spore-forming bacterium, is an important foodborne bacterial pathogen for humans worldwide. Here, we report the complete genome sequence of a Canadian Listeria monocytogenes strain with an antimicrobial resistance (AMR) gene that was isolated from lettuce.

Listeria monocytogenes , a Gram-positive, rod-shaped, non-spore-forming bacterium, is an important foodborne bacterial pathogen for humans worldwide, with a high mortality rate. Here, we report the complete genome sequence of a Listeria monocytogenes strain with an antimicrobial resistance (AMR) gene, isolated from sprouts in Canada.

Listeria monocytogenes is a Gram-positive, rod-shaped, non-spore-forming bacterium which is an important foodborne bacterial pathogen for human worldwide with 20-30% mortality. Here, we report circular complete genome sequences of three Listeria monocytogenes strains isolated from the samples of microgreens in Canada.

Listeria monocytogenes is a Gram-positive, rod-shaped, non-spore-forming bacterium that is an important foodborne bacterial pathogen for humans worldwide, with high mortality rates. Here, we report the complete genome sequence of a Listeria monocytogenes strain that was isolated from kale salad in Canada.

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.

Raoultella planticola is a Gram-negative opportunistic bacterial pathogen associated with hospital-acquired infections in humans. Here, we report the complete genome sequence of one Raoultella planticola strain isolated from Canadian wastewater treatment facilities containing one chromosome and four plasmids with four antimicrobial resistance (AMR) genes and four metal resistance gene clusters.

Despite considerable efforts to control bovine mastitis and explain its causes, it remains the most costly and common disease of dairy cattle worldwide. The role and impact of non-aureus staphylococci (NAS) in udder health are not entirely understood. These Gram-positive bacteria have become the most frequently isolated group of bacteria in milk samples of dairy cows and are associated with (mild) clinical and subclinical mastitis. Different species and strains of NAS differ in their epidemiology, pathogenicity, virulence, ecology and host adaptation, and antimicrobial resistance profiles. They have distinct relationships with the microbiome composition of the udder and may also have protective effects against other mastitis pathogens. Some appear to persist on the skin and in the teat canal and udder, while others seem to be transient residents of the udder from the environment. Analyzing genotypic and phenotypic differences in individual species may also hold clues to why some appear more successful than others in colonizing the udder. Understanding species-level interactions within the microbiome and its interactions with host genetics will clarify the role of NAS in bovine mastitis and udder health.

Salmonella Infantis, a common contaminant of poultry products, is known to harbor mobile genetic elements that confer multi-drug resistance (MDR) and have been detected in many continents. Here, we report four MDR S. Infantis strains recovered from poultry house environments in Santa Cruz Island of the Galapagos showing extended-spectrum β-lactamase (ESBL) resistance and reduced fluoroquinolone susceptibility. Whole-genome sequencing (WGS) revealed the presence of the ESBL-conferring blaCTX-M-65 gene in an IncFIB-like plasmid in three S. Infantis isolates. Multi-locus sequence typing (MLST) and single nucleotide variant/polymorphism (SNP) SNVPhyl analysis showed that the S. Infantis isolates belong to sequence type ST32, likely share a common ancestor, and are closely related (1–3 SNP difference) to blaCTX-M-65-containing clinical and veterinary S. Infantis isolates from the United States and Latin America. Furthermore, phylogenetic analysis of SNPs following core-genome alignment (i.e., ParSNP) inferred close relatedness between the S. Infantis isolates from Galapagos and the United States. Prophage typing confirmed the close relationship among the Galapagos S. Infantis and was useful in distinguishing them from the United States isolates. This is the first report of MDR blaCTX-M-65-containing S. Infantis in the Galapagos Islands and highlights the need for increased monitoring and surveillance programs to determine prevalence, sources, and reservoirs of MDR pathogens.

Despite considerable efforts to control bovine mastitis and explain its causes, it remains the most costly and common disease of dairy cattle worldwide. The role and impact of non-aureus staphylococci (NAS) in udder health are not entirely understood. These Gram-positive bacteria have become the most frequently isolated group of bacteria in milk samples of dairy cows and are associated with (mild) clinical and subclinical mastitis. Different species and strains of NAS differ in their epidemiology, pathogenicity, virulence, ecology and host adaptation, and antimicrobial resistance profiles. They have distinct relationships with the microbiome composition of the udder and may also have protective effects against other mastitis pathogens. Some appear to persist on the skin and in the teat canal and udder, while others seem to be transient residents of the udder from the environment. Analyzing genotypic and phenotypic differences in individual species may also hold clues to why some appear more successful than others in colonizing the udder. Understanding species-level interactions within the microbiome and its interactions with host genetics will clarify the role of NAS in bovine mastitis and udder health.

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., 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.

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.

Complete genome sequences of eight isolates of Salmonella enterica subsp. enterica from Canadian wild birds were determined by MinION and Illumina MiSeq sequencing. Assembled chromosomes had an average size of 4,833,662 bp. Salmonella enterica serovar Worthington obtained from partridge and quail carried 267-kb plasmids, which contained multiple antimicrobial resistance genes.

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.

Background 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. Results Here we provide a comparative analysis of the full genome sequences of 142 prophages of Salmonella enterica subsp. enterica which is the full complement of the prophages that could be retrieved from public databases. We discovered extensive variation in genome sizes (ranging from 6.4 to 358.7 kb) and guanine plus cytosine (GC) content (ranging from 35.5 to 65.4%) and observed a linear correlation between the genome size and the number of open reading frames (ORFs). We used three approaches to compare the phage genomes. The NUCmer/MUMmer genome alignment tool was used to evaluate linkages and correlations based on nucleotide identity between genomes. Multiple sequence alignment was performed to calculate genome average nucleotide identity using the Kalgin program. Finally, genome synteny was explored using dot plot analysis. We found that 90 phage genome sequences grouped into 17 distinct clusters while the remaining 52 genomes showed no close relationships with the other phage genomes and are identified as singletons. We generated genome maps using nucleotide and amino acid sequences which allowed protein-coding genes to be sorted into phamilies (phams) using the Phamerator software. Out of 5796 total assigned phamilies, one phamily was observed to be dominant and was found in 49 prophages, or 34.5% of the 142 phages in our collection. A majority of the phamilies, 4330 out of 5796 (74.7%), occurred in just one prophage underscoring the high degree of diversity among Salmonella bacteriophages. Conclusions Based on nucleotide and amino acid sequences, a high diversity was found among Salmonella bacteriophages which validate the use of prophage sequence analysis as a highly discriminatory subtyping tool for Salmonella. Thorough understanding of the conservation and variation of prophage genomic characteristics will facilitate their rational design and use as tools for bacterial strain construction, vector development and as anti-bacterial 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.

Background 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. Results The antimicrobials resistance varies from as low as 1.9% (2/103) for CTX to as high as 76.7% (79/103) for penicilin in the 103 isolates and 46 (44.7%) S. aureus were determined as multi-resistant isolates with diverse resistance patterns. Thirty-eight virulence gene patterns (with variable frequencies) were identified in the 103 isolates and correlated with MLST types, indicating a great diversity. Although the hla gene also had great diversity (14 genotypes), Hla peptides were relatively more conserved. With 7 clonal complexes identified from 24 spa types and 7 MLST types. Regarding the letter, ST 97 was the dominant type in S. aureus from bovine mastitis in China. Furthermore, based on phylogenetic analysis, there was a distinct evolutionary relationship between the hla gene and MLST. Conclusion Multi-resistant S. aureus occurred in bovine mastitis with diverse resistance patterns. The diversity of virulence gene profiles, especially the hla gene and, their relationship with molecular types were reported for the first time in S. aureus from bovine mastitis, which will be useful for future studies on immunogenicity and vaccine development. In addition, based on the distinct evolutionary relationship between the hla gene and MLST types, we inferred that the hla gene has potential role for molecular typing of S. aureus. Electronic supplementary material The online version of this article (10.1186/s12917-018-1374-7) contains supplementary material, which is available to authorized users.

Background: 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. Results: Comparative analysis of the full genome sequences of 142 prophages of Salmonella enterica subsp. enterica retrieved from public databases revealed an extensive variation in genome sizes (6.4- 358.7 kb) and guanine plus cytosine (GC) content (35.5-65.4%) and a linear correlation between the genome size and the number of open reading frames (ORFs). We used three approaches to compare the phage genomes. The NUCmer/MUMmer genome alignment tool was used to evaluate linkages and correlations based on nucleotide identity between genomes. Multiple sequence alignment was performed to calculate genome average nucleotide identity using the Kalgin program. Finally, genome synteny was explored using dot plot analysis. We found that 90 phage genome sequences grouped into 17 distinct clusters while the remaining 52 genomes showed no close relationships with the other phage genomes and are identified as singletons. We generated genome maps using nucleotide and amino acid sequences which allowed protein-coding genes to be sorted into phamilies (phams) using the Phamerator software. Out of 5796 total assigned phamilies, one phamily was observed to be dominant and was found in 49 prophages, or 34.5% of the 142 phages in our collection. A majority of the phamilies, 4330 out of 5796 (74.7%), occurred in just one prophage underscoring the high degree of diversity among Salmonella bacteriophages. Conclusions: Based on nucleotide and amino acid sequences, a high diversity was found among Salmonella bacteriophages which validate the use of prophage sequence analysis as a highly discriminatory subtyping tool for Salmonella. Thorough understanding of the conservation and variation of prophage genomic characteristics will facilitate their rational design and use as tools for bacterial strain construction, vector development and as anti-bacterial agents.

A novel type strain, designated SDB 2975T (=CECT 9737T=DSM 105892T), of the novel species Staphylococcus debuckii sp. nov. isolated from bovine milk is described. The novel species belongs to the genus Staphylococcus and showed resistance to tetracycline and was oxidase- and coagulase-negative, catalase-positive, and Gram-stain-positive. Phylogenetic relationships of Staphylococcus debuckii SDB 2975T to other staphylococcal species were inferred from 16S rRNA gene and whole-genome-based phylogenetic reconstruction. The 16S rRNA gene comparisons showed that the strain is closely related to Staphylococcus condimenti (99.73 %), Staphylococcus piscifermentans (99.66 %), Staphylococcus carnosus (99.59 %) and Staphylococcus simulans (98.03 %). Average nucleotide identity (ANI) values between S.taphylococcus debuckii SDB 2975T and its closely related Staphylococcus species were 83.96, 94.5, 84.03 and 78.09 %, respectively, and digital DNA–DNA hybridization (dDDH) values were 27.70, 58.02, 27.70 and 22.00 %, respectively. The genome of Staphylococcus debuckii SDB 2975T was sequenced with PacBio and Illumina technologies and is 2 691 850 bp long, has a G+C content of 36.6 mol% and contains 2678 genes and 80 RNAs, including six copies of each5S rRNA, 16S rRNA and 23S rRNA genes. Biochemical profiling and a newly developed PCR assay enabled differentiation of Staphylococcus debuckii SDB 2975T and three other SDB strains from its closest staphylococcal species. Differentiation was also achieved by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF). Genes unique to Staphylococcus debuckii were identified and a PCR-based assay was developed to differentiate Staphylococcus debuckii from other staphylococcal species. In conclusion, the results of phylogenetic analysis along with the ANI values

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., 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.

TheHalobacteriaconstitute one of the largest groups within theArchaea. The hierarchical relationship among members of this large class, which comprises a single order and a single family, has proven difficult to determine based upon 16S rRNA gene trees and morphological and physiological characteristics. This work reports detailed phylogenetic and comparative genomic studies on >100 halobacterial (haloarchaeal) genomes containing representatives from 30 genera to investigate their evolutionary relationships. In phylogenetic trees reconstructed on the basis of 32 conserved proteins, using both neighbour-joining and maximum-likelihood methods, two major clades (clades A and B) encompassing nearly two-thirds of the sequenced haloarchaeal species were strongly supported. Clades grouping the same species/genera were also supported by the 16S rRNA gene trees and trees for several individual highly conserved proteins (RpoC, EF-Tu, UvrD, GyrA, EF-2/EF-G). In parallel, our comparative analyses of protein sequences from haloarchaeal genomes have identified numerous discrete molecular markers in the form of conserved signature indels (CSI) in protein sequences and conserved signature proteins (CSPs) that are found uniquely in specific groups of haloarchaea. Thirteen CSIs in proteins involved in diverse functions and 68 CSPs that are uniquely present in all or most genome-sequenced haloarchaea provide novel molecular means for distinguishing members of the classHalobacteriafrom all other prokaryotes. The members of clade A are distinguished from all other haloarchaea by the unique shared presence of two CSIs in the ribose operon protein and small GTP-binding protein and eight CSPs that are found specifically in members of this clade. Likewise, four CSIs in different proteins and five other CSPs are present uniquely in members of clade B and distinguish them from all other haloarchaea. Based upon their specific clustering in phylogenetic trees for different gene/protein sequences and the unique shared presence of large numbers of molecular signatures, members of clades A and B are indicated to be distinct from all other haloarchaea because of their uniquely shared evolutionary histories. Based upon these results, it is proposed that clades A and B be recognized as two new orders,Natrialbalesord. nov. andHaloferacalesord. nov., within the classHalobacteria, containing the novel familiesNatrialbaceaefam. nov. andHaloferacaceaefam. nov. Other members of the classHalobacteriathat are not members of these two orders will remain part of the emended orderHalobacterialesin an emended familyHalobacteriaceae.

The current taxonomy of the order Xanthomonadales is highly problematic and no comprehensive phylogenomic studies have been completed that include the most divergent members within the order. In this work, we have completed a phylogenomic analysis of a wide range of genomes, five of which were sequenced for the first time for this work, representing the vast majority of the diversity within the order Xanthomonadales. Using comparative genomic techniques, we have identified a large number of conserved signature inserts/deletions (CSIs) that are specifically found in different groups of related organisms, at different taxonomic levels, within the order. Our phylogenetic analyses do not support a monophyletic grouping of the members of the order Xanthomonadales and no CSIs were identified which are uniquely shared by all sequenced species within this order. However, our work has identified 10 CSIs which are specific to all members of the family Xanthomonadaceae and an additional 10 and 11 CSIs that are specific to one of two phylogenetically well-defined clades within the family Xanthomonadaceae. On the basis of the identified CSIs and the results of phylogenomic analyses, we propose a new taxonomic framework for the order Xanthomonadales. In this proposal, the families Algiphilaceae and Solimonadaceae (Nevskiaceae), which do not branch with the other members of the order Xanthomonadales, are transferred into the order Nevskiales ord. nov. The remaining members of the order Xanthomonadales are divided into two families: the family Xanthomonadaceae, containing the genus Xanthomonas and its closest relatives, and a new family, Rhodanobacteraceae fam. nov., containing the genus Rhodanobacter and its closest relatives. Additionally, we have also emended descriptions of the order Lysobacterales, the family Lysobacteraceae, and the family Nevskiaceae to indicate that they are earlier synonyms of the order Xanthomonadales, the family Xanthomonadaceae, and the family Solimonadaceae, respectively.

Non- aureus staphylococci (NAS), the bacteria most commonly isolated from the bovine udder, potentially protect the udder against infection by major mastitis pathogens due to bacteriocin production. In this study, we determined the inhibitory capability of 441 bovine NAS isolates (comprising 26 species) against bovine Staphylococcus aureus . Furthermore, inhibiting isolates were tested against a human methicillin-resistant S. aureus (MRSA) isolate using a cross-streaking method. We determined the presence of bacteriocin clusters in NAS whole genomes using genome mining tools, BLAST, and comparison of genomes of closely related inhibiting and noninhibiting isolates and determined the genetic organization of any identified bacteriocin biosynthetic gene clusters. Forty isolates from 9 species ( S. capitis , S. chromogenes , S. epidermidis , S. pasteuri , S. saprophyticus , S. sciuri , S. simulans , S. warneri , and S. xylosus ) inhibited growth of S. aureus in vitro , 23 isolates of which, from S. capitis , S. chromogenes , S. epidermidis , S. pasteuri , S. simulans , and S. xylosus , also inhibited MRSA. One hundred five putative bacteriocin gene clusters encompassing 6 different classes (lanthipeptides, sactipeptides, lasso peptides, class IIa, class IIc, and class IId) in 95 whole genomes from 16 species were identified. A total of 25 novel bacteriocin precursors were described. In conclusion, NAS from bovine mammary glands are a source of potential bacteriocins, with >21% being possible producers, representing potential for future characterization and prospective clinical applications. IMPORTANCE Mastitis (particularly infections caused by Staphylococcus aureus ) costs Canadian dairy producers $400 million/year and is the leading cause of antibiotic use on dairy farms. With increasing antibiotic resistance and regulations regarding use, there is impetus to explore bacteriocins (bacterially produced antimicrobial peptides) for treatment and prevention of bacterial infections. We examined the ability of 441 NAS bacteria from Canadian bovine milk samples to inhibit growth of S. aureus in the laboratory. Overall, 9% inhibited growth of S. aureus and 58% of those also inhibited MRSA. In NAS whole-genome sequences, we identified >21% of NAS as having bacteriocin genes. Our study represents a foundation to further explore NAS bacteriocins for clinical use.

Assessment of antimicrobial use (AMU) is vital for interpreting the origin of changes in antimicrobial resistance (AMR). The objectives of the present study were to estimate the association between AMU determined using on-farm treatment records (TR) and inventory of empty drug containers (INV). Herds were selected to represent Canadian dairy farms. Producers were asked to record animal health events and treatments on a standard General Health Event form. For inventory data, 40-L receptacles were placed at various locations considered convenient to deposit all empty drug containers. Antimicrobial defined-daily dosages (ADD) were calculated for 51 Canadian herds using the 2 methods. Estimation of AMU was 31,840 ADD using the INV and 14,487 ADD using the TR, indicating that for every TR entry, 2.20 times more treatments were observed using the INV. Mastitis, reproductive conditions, and dry cow therapy were the most frequent reasons for antimicrobial therapy when assessing TR. For all antimicrobials evaluated, mean ADD was higher using the INV versus TR. Regardless, a strong positive correlation (0.80) was observed between the 2 methods, indicating that herds with increased number of ADD recorded using the INV also had increased number of ADD recorded using TR. Furthermore, a positive association was observed for the 6 most commonly used antimicrobials. In comparison to methods used in surveillance programs on AMU in livestock that assume a constant use in all herds (i.e., sales data), INV provided a herd-level specific quantity of AMU positively correlated with AMU recorded at the animal level in general. The INV was easy to implement and provided a measure of total AMU in the herd. Availability of such information would be valuable for interpreting changes in AMR at the herd level and enabling evaluation of interventions for decreasing AMR.

Non-aureus staphylococci (NAS), the microorganisms most frequently isolated from bovine milk worldwide, are a heterogeneous group of numerous species. To establish their importance as a group, the distribution of individual species needs to be determined. In the present study, NAS intramammary infection (IMI) was defined as a milk sample containing ≥1,000 cfu/mL in pure or mixed culture that was obtained from a cohort of cows assembled by the Canadian Bovine Mastitis Research Network. Overall, 6,213 (6.3%) of 98,233 quarter-milk samples from 5,149 cows and 20,305 udder quarters were associated with an NAS IMI. Of the 6,213 phenotypically identified NAS isolates, 5,509 (89%) were stored by the Canadian Bovine Mastitis Research Network Mastitis Pathogen Collection and characterized using partial sequencing of the rpoB housekeeping gene, confirming 5,434 isolates as NAS. Prevalence of each NAS species IMI was estimated using Bayesian models, with presence of a specific NAS species as the outcome. Overall quarter-level NAS IMI prevalence was 26%. The most prevalent species causing IMI were Staphylococcus chromogenes (13%), Staphylococcus simulans (4%), Staphylococcus haemolyticus (3%), Staphylococcus xylosus (2%), and Staphylococcus epidermidis (1%). The prevalence of NAS IMI as a group was highest in first-parity heifers and was evenly distributed throughout cows in parities ≥2. The IMI prevalence of some species such as S. chromogenes, S. simulans, and S. epidermidis differed among parities. Overall prevalence of NAS IMI was 35% at calving, decreased over the next 10 d, and then gradually increased until the end of lactation. The prevalence of S. chromogenes, Staphylococcus gallinarum, Staphylococcus cohnii, and Staphylococcus capitis was highest at calving, whereas the prevalence of S. chromogenes, S. haemolyticus, S. xylosus, and S. cohnii increased during lactation. Although the overall prevalence of NAS IMI was similar across barn types, the prevalence of S. simulans, S. xylosus, S. cohnii, Staphylococcus saprophyticus, S. capitis, and Staphylococcus arlettae IMI was higher in tiestall barns; the prevalence of S. epidermidis IMI was lowest; and the prevalence of S. chromogenes and Staphylococcus sciuri IMI was highest in bedded-pack barns. Staphylococcus simulans, S. epidermidis, S. xylosus, and S. cohnii IMI were more prevalent in herds with intermediate to high bulk milk somatic cell count (BMSCC) and S. haemolyticus IMI was more prevalent in herds with high BMSCC, whereas other common NAS species IMI were equally prevalent in all 3 BMSCC categories. Distribution of NAS species IMI differed among the 4 regions of Canada. In conclusion, distribution differed considerably among NAS species IMI; therefore, accurate identification (species level) is essential for studying NAS epidemiology.

The members of the order Pasteurellales are currently distinguished primarily on the basis of their branching in the rRNA trees and no convincing biochemical or molecular markers are known that distinguish them from all other bacteria. The genome sequences for 20 Pasteurellaceae species/strains are now publicly available. We report here detailed analyses of protein sequences from these genomes to identify conserved signature indels (CSIs) that are specific for either all Pasteurellales or its major clades. We describe more than 23 CSIs in widely distributed genes/proteins that are uniquely shared by all sequenced Pasteurellaceae species/strains but are not found in any other bacteria. Twenty-one additional CSIs are also specific for the Pasteurellales except in some of these cases homologues were not detected in a few species or the CSI was also present in an isolated non-Pasteurellaceae species. The sequenced Pasteurellaceae species formed two distinct clades in a phylogenetic tree based upon concatenated sequences for 10 conserved proteins. The first of these clades consisting of Aggregatibacter, Pasteurella, Actinobacillus succinogenes, Mannheimia succiniciproducens, Haemophilus influenzae and Haemophilus somnus was also independently supported by 13 uniquely shared CSIs that are not present in other Pasteurellaceae species or other bacteria. Another clade consisting of the remaining Pasteurellaceae species (viz. Actinobacillus pleuropneumoniae, Actinobacillus minor, Haemophilus ducryi, Mannheimia haemolytica and Haemophilus parasuis) was also strongly and independently supported by nine CSIs that are uniquely present in these bacteria. The order Pasteurellales is presently made up of a single family, Pasteurellaceae, that encompasses all of its genera. In this context, our identification of two distinct clades within the Pasteurellales, which are supported by both phylogenetic analyses and by multiple highly specific molecular markers, strongly argues for and provides potential means for the division of various genera from this order into a minimum of two families. The genetic changes responsible for these CSIs were likely introduced in the common ancestors of either all Pasteurellales or of these two specific clades. These CSIs provide novel means for the identification and circumscription of these groups of Pasteurellales in molecular terms.

The evolutionary interrelationships between the archaeal organisms which comprise the class Halobacteria have proven difficult to elucidate using traditional phylogenetic tools. The class currently contains three orders. However, little is known about the family level relationships within these orders. In this work, we have completed a comprehensive comparative analysis of 129 sequenced genomes from members of the class Halobacteria in order to identify shared molecular characteristics, in the forms of conserved signature insertions/deletions (CSIs) and conserved signature proteins (CSPs), which can provide reliable evidence, independent of phylogenetic trees, that the species from the groups in which they are found are specifically related to each other due to common ancestry. Here we present 20 CSIs and 31 CSPs which are unique characteristics of infra-order level groups of genera within the class Halobacteria. We also present 40 CSIs and 234 CSPs which are characteristic of Haloarcula, Halococcus, Haloferax, or Halorubrum. Importantly, the CSIs and CSPs identified here provide evidence that the order Haloferacales contains two main groups, one consisting of Haloferax and related genera supported by four CSIs and five CSPs and the other consisting of Halorubrum and related genera supported by four CSPs. We have also identified molecular characteristics that suggest that the polyphyletic order Halobacteriales contains at least two large monophyletic clusters of organisms in addition to the polyphyletic members of the order, one cluster consisting of Haloarcula and related genera supported by ten CSIs and nineteen CSPs and the other group consisting of the members of the genus Halococcus supported by nine CSIs and 23 CSPs. We have also produced a highly robust phylogenetic tree based on the concatenated sequences of 766 proteins which provide additional support for the relationships identified by the CSIs and CSPs. On the basis of the phylogenetic analyses and the identified conserved molecular characteristics presented here, we propose a division of the order Haloferacales into two families, an emended family Haloferacaceae and Halorubraceae fam. nov. and a division of the order Halobacteriales into three families, an emended family Halobacteriaceae, Haloarculaceae fam. nov., and Halococcaceae fam. nov.

The phylum Chlamydiae contains nine ecologically and genetically diverse families all placed within a single order. In this work, we have completed a comprehensive comparative analysis of 36 sequenced Chlamydiae genomes in order to identify shared molecular characteristics, namely conserved signature insertions/deletions (CSIs) and conserved signature proteins (CSPs), which can serve as distinguishing characteristics of supra-familial clusters within the phylum Chlamydiae. Our analysis has led to the identification of 32 CSIs which are specific to clusters within the phylum Chlamydiae at various phylogenetic depths. Importantly, 17 CSIs and 98 CSPs were found to be specific for the family Chlamydiaceae while another 3 CSI variants and 15 CSPs were specific for a grouping of the families Criblamydiaceae, Parachlamydiaceae, Simkaniaceae and Waddliaceae. These two clusters were also found to be distinguishable in 16S rRNA based phylogenetic trees, concatenated protein based phylogenetic trees, character compatibility based phylogenetic analyses, and on the basis of 16S rRNA gene sequence identity and average amino acid identity values. On the basis of the identified molecular characteristics, branching in phylogenetic trees, and the genetic distance between the two clusters within the phylum Chlamydiae we propose a division of the class Chlamydiia into two orders: an emended order Chlamydiales, containing the family Chlamydiaceae and the closely related Candidatus family Clavichlamydiaceae, and the novel order Parachlamydiales ord. nov. containing the families Parachlamydiaceae, Simkaniaceae and Waddliaceae and the Candidatus families Criblamydiaceae, Parilichlamydiaceae, Piscichlamydiaceae, and Rhabdochlamydiaceae. We also include a brief discussion of the reunification of the genera Chlamydia and Chlamydophila.

The generaActinobacillus, Haemophilus,andPasteurellaexhibit 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 ofActinobacillus, Haemophilus,andPasteurellaspecies which represent the “sensu stricto” members of these genera. We have identified 3, 7, and 6 conserved signature indels (CSIs), which are specifically shared bysensu strictomembers ofActinobacillus, Haemophilus,andPasteurella, respectively. We have also identified two different sets of CSIs that are unique characteristics of the pathogen containing generaAggregatibacterandMannheimia, respectively. It is now possible to demarcate the generaActinobacillus sensu stricto, Haemophilus sensu stricto,andPasteurella sensu strictoon the basis of discrete molecular signatures. The other members of the generaActinobacillus, Haemophilus,andPasteurellathat 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.

Genome sequences are enabling applications of different approaches to more clearly understand microbial phylogeny and systematics. Two of these approaches involve identification of conserved signature indels (CSIs) and conserved signature proteins (CSPs) that are specific for different lineages. These molecular markers provide novel and more definitive means for demarcation of prokaryotic taxa and for identification of species from these groups. Genome sequences are also enabling determination of phylogenetic relationships among species based upon sequences for multiple proteins. In this work, we have used all of these approaches for studying the phytopathogenic bacteria belonging to the genera Dickeya , Pectobacterium and Brenneria . Members of these genera, which cause numerous diseases in important food crops and ornamental plants, are presently distinguished mainly on the basis of their branching in phylogenetic trees. No biochemical or molecular characteristic is known that is uniquely shared by species from these genera. Hence, detailed studies using the above approaches were carried out on proteins from the genomes of these bacteria to identify molecular markers that are specific for them. In phylogenetic trees based upon concatenated sequences for 23 conserved proteins, members of the genera Dickeya , Pectobacterium and Brenneria formed a strongly supported clade within the other Enterobacteriales . Comparative analysis of protein sequences from the Dickeya , Pectobacterium and Brenneria genomes has identified 10 CSIs and five CSPs that are either uniquely or largely found in all genome-sequenced species from these genera, but not present in any other bacteria in the database. In addition, our analyses have identified 10 CSIs and 17 CSPs that are specifically present in either all or most sequenced Dickeya species/strains, and six CSIs and 19 CSPs that are uniquely found in the sequenced Pectobacterium genomes. Finally, our analysis also identified three CSIs and one CSP that are specifically shared by members of the genera Pectobacterium and Brenneria , but absent in species of the genus Dickeya , indicating that the former two genera shared a common ancestor exclusive of Dickeya . The identified CSIs and CSPs provide novel tools for identification of members of the genera Dickeya and Pectobacterium and for delimiting these taxa in molecular terms. Descriptions of the genera Dickeya and Pectobacterium have been revised to provide information for these molecular markers. Biochemical studies on these CSIs and CSPs, which are specific for these genera, may lead to discovery of novel properties that are unique to these bacteria and which could be targeted to develop antibacterial agents that are specific for these plant-pathogenic bacteria.

The species from the order Xanthomonadales, which harbors many important plant pathogens and some human pathogens, are currently distinguished primarily on the basis of their branching in the 16S rRNA tree. No molecular or biochemical characteristic is known that is specific for these bacteria. Phylogenetic and comparative analyses were conducted on 26 sequenced Xanthomonadales genomes to delineate their branching order and to identify molecular signatures consisting of conserved signature indels (CSIs) in protein sequences that are specific for these bacteria. In a phylogenetic tree based upon sequences for 28 proteins, Xanthomonadales species formed a strongly supported clade with Rhodanobacter sp. 2APBS1 as its deepest branch. Comparative analyses of protein sequences have identified 13 CSIs in widely distributed proteins such as GlnRS, TypA, MscL, LysRS, LipA, Tgt, LpxA, TolQ, ParE, PolA and TyrB that are unique to all species/strains from this order, but not found in any other bacteria. Fifteen additional CSIs in proteins (viz. CoxD, DnaE, PolA, SucA, AsnB, RecA, PyrG, LigA, MutS and TrmD) are uniquely shared by different Xanthomonadales except Rhodanobacter and in a few cases by Pseudoxanthomonas species, providing further support for the deep branching of these two genera. Five other CSIs are commonly shared by Xanthomonadales and 1-3 species from the orders Chromatiales, Methylococcales and Cardiobacteriales suggesting that these deep branching orders of Gammaproteobacteria might be specifically related. Lastly, 7 CSIs in ValRS, CarB, PyrE, GlyS, RnhB, MinD and X001065 are commonly shared by Xanthomonadales and a limited number of Beta- or Gamma-proteobacteria. Our analysis indicates that these CSIs have likely originated independently and they are not due to lateral gene transfers. The Xanthomonadales-specific CSIs reported here provide novel molecular markers for the identification of these important plant and human pathogens and also as potential targets for development of drugs/agents that specifically target these bacteria.

The analyses of genome sequences have led to the proposal that lateral gene transfers (LGTs) among prokaryotes are so widespread that they disguise the interrelationships among these organisms. This has led to questioning of whether the Darwinian model of evolution is applicable to prokaryotic organisms. In this review, we discuss the usefulness of taxon-specific molecular markers such as conserved signature indels (CSIs) and conserved signature proteins (CSPs) for understanding the evolutionary relationships among prokaryotes and to assess the influence of LGTs on prokaryotic evolution. The analyses of genomic sequences have identified large numbers of CSIs and CSPs that are unique properties of different groups of prokaryotes ranging from phylum to genus levels. The species distribution patterns of these molecular signatures strongly support a tree-like vertical inheritance of the genes containing these molecular signatures that is consistent with phylogenetic trees. Recent detailed studies in this regard on the Thermotogae and Archaea, which are reviewed here, have identified large numbers of CSIs and CSPs that are specific for the species from these two taxa and a number of their major clades. The genetic changes responsible for these CSIs (and CSPs) initially likely occurred in the common ancestors of these taxa and then vertically transferred to various descendants. Although some CSIs and CSPs in unrelated groups of prokaryotes were identified, their small numbers and random occurrence has no apparent influence on the consistent tree-like branching pattern emerging from other markers. These results provide evidence that although LGT is an important evolutionary force, it does not mask the tree-like branching pattern of prokaryotes or understanding of their evolutionary relationships. The identified CSIs and CSPs also provide novel and highly specific means for identification of different groups of microbes and for taxonomical and biochemical studies.

The PVC superphylum is an amalgamation of species from the phyla Planctomycetes, Verrucomicrobia, and Chlamydiae, along with the Lentisphaerae, Poribacteria, and two other candidate divisions. The diverse species of this superphylum lack any significant marker that differentiates them from other bacteria. Recently, genome sequences for 37 species covering all of the main PVC groups of bacteria have become available. We have used these sequences to construct a phylogenetic tree based upon concatenated sequences for 16 proteins and identify molecular signatures in protein sequences that are specific for the species from these phyla or those providing molecular links among them. Of the useful molecular markers identified in the present work, six conserved signature indels (CSIs) in the proteins Cyt c oxidase, UvrD helicase, urease, and a helicase-domain containing protein are specific for the species from the Verrucomicrobia phylum; three other CSIs in an ABC transporter protein, cobyrinic acid ac-diamide synthase, and SpoVG protein are specific for the Planctomycetes species. Additionally, a 3 aa insert in the RpoB protein is uniquely present in all sequenced Chlamydiae, Verrucomicrobia, and Lentisphaerae species, providing evidence for the shared ancestry of the species from these three phyla. Lastly, we have also identified a conserved protein of unknown function that is exclusively found in all sequenced species from the phyla Chlamydiae, Verrucomicrobia, Lentisphaerae, and Planctomycetes suggesting a specific linkage among them. The absence of this protein in Poribacteria, which branches separately from other members of the PVC clade, indicates that it is not specifically related to the PVC clade of bacteria. The molecular markers described here in addition to clarifying the evolutionary relationships among the PVC clade of bacteria also provide novel tools for their identification and for genetic and biochemical studies on these organisms.