Comparative genomic analyses provide insight into the pathogenicity of three Pseudomonas syringae pv. actinidiae strains from Anhui Province, China.

BACKGROUND: Pseudomonas syringae pv. actinidiae (Psa) is an important bacterial plant pathogen that causes severe damage to the kiwifruit industry worldwide. Three Psa strains were recently obtained from different kiwifruit orchards in Anhui Province, China. The present study mainly focused on the variations in virulence and genome characteristics of these strains based on the pathogenicity assays and comparative genomic analyses. RESULTS: Three strains were identified as biovar 3 (Psa3), along with strain QSY6 showing higher virulence than JZY2 and YXH1 in pathogenicity assays. The whole genome assembly revealed that each of the three strains had a circular chromosome and a complete plasmid. The chromosome sizes ranged from 6.5 to 6.6 Mb with a GC content of approximately 58.39 to 58.46%, and a predicted number of protein-coding sequences ranging from 5,884 to 6,019. The three strains clustered tightly with 8 Psa3 reference strains in terms of average nucleotide identity (ANI), whole-genome-based phylogenetic analysis, and pangenome analysis, while they were evolutionarily distinct from other biovars (Psa1 and Psa5). Variations were observed in the repertoire of effectors of the type III secretion system among all 15 strains. Moreover, synteny analysis of the three sequenced strains revealed eight genomic regions containing 308 genes exclusively present in the highly virulent strain QSY6. Further investigation of these genes showed that 16 virulence-related genes highlight several key factors, such as effector delivery systems (type III secretion systems) and adherence (type IV pilus), which might be crucial for the virulence of QSY6. CONCLUSION: Three Psa strains were identified and showed variant virulence in kiwifruit plant. Complete genome sequences and comparative genomic analyses further provided a theoretical basis for the potential pathogenic factors responsible for kiwifruit bacterial canker.

Phylogenomic and molecular marker based studies to clarify the evolutionary relationships amongst Anoxybacillus species and demarcation of the family Anoxybacillaceae and some of its constituent genera.

The family Anoxybacillaceae was recently proposed encompassing the genera Anoxybacillus, Geobacillus, Parageobacillus, Saccharococcus and Thermolongibacillus. Of these genera, Anoxybacillus contains >50% of the Anoxybacillaceae species. However, Anoxybacillus species form multiple unrelated clades in phylogenetic trees and their evolutionary relationships are unclear. To clarify the evolutionary relationships of Anoxybacillus and other Anoxybacillaceae species, detailed phylogenomic and comparative analyses were conducted on 38 Anoxybacillaceae species with available genomes. In a phylogenomic tree based on 1148 core proteins, all Anoxybacillus, Geobacillus, Parageobacillus, Saccharococcus and Thermolongibacillus species, excepting Anoxybacillus sediminis, formed a strongly supported clade representing the family Anoxybacillaceae. Five conserved signature indels (CSIs) reported here are also uniquely found in these species, providing robust means for the demarcation of family Anoxybacillaceae in molecular terms. In our phylogenomic tree and in the Genomic Taxonomy Database, Anoxybacillus species formed four distinct clades designated as Anoxybacillus sensu stricto (containing the type species A. pushchinoensis), Anoxybacillus_A, Anoxybacillus_B and Anoxybacillus_C. Our analyses have identified 17 novel CSIs which offer means to reliably distinguish species from these clades based upon multiple uniquely shared molecular characteristics. Additionally, we have identified three and seven CSIs specific for the genera Geobacillus and Brevibacillus, respectively. All seven Brevibacillus-specific CSIs are also shared by Anoxybacillus sediminis, which branches reliably with this genus. Based on the strong phylogenetic and molecular evidence presented here, we are proposing that the genus Anoxybacillus should be restricted to only the species from Anoxybacillus sensu stricto clade, whereas the species from Anoxybacillus_A, Anoxybacillus_B, and Anoxybacillus_C clades should be transferred into three novel genera Anoxybacteroides gen. nov., Paranoxybacillus gen. nov. and Thermaerobacillus gen. nov., respectively. Additionally, we are also proposing the transfer of Anoxybacillus sediminis to the genus Brevibacillus. The proposed changes, which reliably depict the evolutionary relationships among Anoxybacillaceae species, should be helpful in the studies of these organisms.

Robust demarcation of the family Peptostreptococcaceae and its main genera based on phylogenomic studies and taxon-specific molecular markers.

The family Peptostreptococcaceae, which contains 15 genera including Clostridioides, presently lacks proper circumscription. Using 52 available genomes for Peptostreptococcaceae species, we report comprehensive phylogenomic and comparative analyses to reliably discern their evolutionary relationships. In phylogenetic trees based on core genome proteins and 16S rRNA gene sequences, the examined species formed a strongly supported clade designated as Peptostreptococcaceae sensu stricto. This clade encompassed the genera Peptostreptococcus (type genus), Asaccharospora, Clostridioides, Intestinibacter, Paeniclostridium, Paraclostridium, Peptacetobacter, Romboutsia and Terrisporobacter, and two misclassified species (viz. Eubacterium tenue and 'Clostridium dakarense'). The distinctness of this clade is strongly supported by eight identified conserved signature indels (CSIs), which are specific for the species from this clade. Based on the robust evidence provided by presented studies, we are proposing the emendment of family Peptostreptococcaceae to only the genera within the Peptostreptococcaceae sensu stricto clade. We also report 67 other novel CSIs, which reliably demarcate different Peptostreptococcaceae species clades and clarify the classification of some misclassified species. Based on the consistent evidence obtained from different presented studies, we are making the following proposals to clarify the classification of Peptostreptococcaceae species: (i) transfer of Eubacterium tenue, Paeniclostridium ghonii and Paeniclostridium sordellii as comb. nov. into the genus Paraclostridium; (ii) transfer of Clostridioides mangenotii as a comb. nov. into Metaclostridioides gen. nov.; (iii) classification of 'Clostridium dakarense' as a novel species Faecalimicrobium dakarense gen. nov., sp. nov. (type strain FF1T; genome and 16S rRNA accession numbers GCA_000499525.1 and KC517358, respectively); (iv) transfer of two misclassified species, Clostridium paradoxum and Clostridium thermoalcaliphilum, into Alkalithermobacter gen. nov.; and (v) proposals for two novel families, Peptoclostridiaceae fam. nov. and Tepidibacteraceae fam. nov., to accommodate remaining unclassified Peptostreptococcaceae genera. The described CSIs specific for different families and genera provide novel and reliable means for the identification, diagnostics and biochemical studies on these bacteria.

Phylogenomic and molecular markers based studies on Staphylococcaceae and Gemella species. Proposals for an emended family Staphylococcaceae and three new families (Abyssicoccaceae fam. nov., Salinicoccaceae fam. nov. and Gemellaceae fam. nov.) harboring four new genera, Lacicoccus gen. nov., Macrococcoides gen. nov., Gemelliphila gen. nov., and Phocicoccus gen. nov.

The family Staphylococcacae and genus Gemella contain several organisms of clinical or biotechnological importance. We report here comprehensive phylogenomic and comparative analyses on 112 available genomes from species in these taxa to clarify their evolutionary relationships and classification. In a phylogenomic tree based on 678 core proteins, Gemella species were separated from Staphylococcacae by a long branch indicating that they constitute a distinct family (Gemellaceae fam. nov.). In this tree, Staphylococcacae species formed two main clades, one encompassing the genera Aliicoccus, Jeotgalicoccus, Nosocomiicoccus and Salinicoccus (Family "Salinicoccaceae"), while the other clade consisted of the genera Macrococcus, Mammaliicoccus and Staphylococcus (Family Staphylococcaceae emend.). In this tree, species from the genera Gemella, Jeotgalicoccus, Macrococcus and Salinicoccus each formed two distinct clades. Two species clades for these genera are also observed in 16S rRNA gene trees and supported by average amino acid identity analysis. We also report here detailed analyses on protein sequences from Staphylococcaceae and Gemella genomes to identify conserved signature indels (CSIs) which are specific for different genus and family-level clades. These analyses have identified 120 novel CSIs robustly demarcating different proposed families and genera. The identified CSIs provide independent evidence that the genera Gemella, Jeotgalicoccus, Macrococcus and Salinicoccus consist of two distinct clades, which can be reliably distinguished based on multiple exclusively shared CSIs. We are proposing transfers of the species from the novel clades of the above four genera into the genera Gemelliphila gen. nov., Phocicoccus gen. nov., Macrococcoides gen. nov. and Lacicoccus gen. nov., respectively. The identified CSIs also provide strong evidence for division of Staphylococcaceae into an emended family Staphylococcaceae and two new families, Abyssicoccaceae fam. nov. and Salinicoccaceae fam. nov. All of these families can be reliably demarcated based on several exclusively shared CSIs.

Phylogenomic and comparative genomic analyses of species of the family Pseudomonadaceae: Proposals for the genera Halopseudomonas gen. nov. and Atopomonas gen. nov., merger of the genus Oblitimonas with the genus Thiopseudomonas, and transfer of some misclassified species of the genus Pseudomonas into other genera.

The evolutionary relationships among species of the family Pseudomonadaceae were examined based on 255 available genomes representing >85 % of the species from this family. In a phylogenetic tree based on concatenated sequences of 118 core proteins, most species of the genus Pseudomonas grouped within one large cluster which also included members of the genera Azotobacter and Azomonas. Within this large cluster 18-30 clades/subclades of species of the genus Pseudomonas consisting of between 1 and 36 species, were observed. However, a number of species of the genus Pseudomonas branched outside of this main cluster and were interspersed among other genera of the family Pseudomonadaceae. This included a strongly supported clade (Pertucinogena clade) consisting of 19 mainly halotolerant species. The distinctness of this clade from all other members of the family Pseudomonadaceae is strongly supported by 24 conserved signature indels (CSIs) in diverse proteins that are exclusively found in all members of this clade. Nine uncharacterized members of the genus Pseudomonas also shared these CSIs and they branched within the Pertucinogena clade, indicating their affiliation to this clade. On the basis of the strong evidence supporting the distinctness of the Pertucinogena clade, we are proposing transfer of species from this clade into a novel genus Halopseudomonas gen. nov. Pseudomonas caeni also branches outside of the main cluster and groups reliably with Oblitimonas alkaliphila and Thiopseudomonas denitrificans. Six identified CSIs are uniquely shared by these three species and we are proposing their integration into the emended genus Thiopseudomonas, which has priority over the name Oblitimonas. We are also proposing transfer of the deep-branching Pseudomonas hussainii, for which 22 exclusive CSIs have been identified, into the genus Atopomonas gen. nov. Lastly, we present strong evidence that the species Pseudomonas cissicola and Pseudomonas geniculata are misclassified into the genus Pseudomonas and that they are specifically related to the genera Xanthomonas and Stenotrophomonas, respectively. In addition, we are also reclassifying 'Pseudomonas acidophila' as Paraburkholderia acidicola sp. nov. (Type strain: G-6302=ATCC 31363=BCRC 13035).

A robust phylogenetic framework for members of the order Legionellales and its main genera (Legionella, Aquicella, Coxiella and Rickettsiella) based on phylogenomic analyses and identification of molecular markers demarcating different clades.

The order Legionellales contains several clinically important microorganisms. Although members of this order are well-studied for their pathogenesis, there is a paucity of reliable characteristics distinguishing members of this order and its constituent genera. Genome sequences are now available for 73 Legionellales species encompassing ≈90% of known members from different genera. With the aim of understanding evolutionary relationships and identifying reliable molecular characteristics that are specific for this order and its constituent genera, detailed phylogenetic and comparative analyses were conducted on the protein sequences from these genomes. A phylogenomic tree was constructed based on 393 single copy proteins that are commonly shared by the members of this order to delineate the evolutionary relationships among its members. In parallel, comparative analyses were performed on protein sequences from Legionellales genomes to identify novel molecular markers consisting of conserved signature indels (CSIs) that are specific for different clades and genera. In the phylogenomic tree and in an amino acid identity matrix based on core proteins, members of the genera Aquicella, Coxiella, Legionella and Rickettsiella formed distinct clades confirming their monophyly. In these studies, Diplorickettsia massiliensis exhibited a close relationship to members of the genus Rickettsiella. The results of our comparative genomic analyses have identified 59 highly specific molecular markers consisting of CSIs in diverse proteins that are uniquely shared by different members of this order. Four of these CSIs are specific for all Legionellales species, except the two deeper-branching "Candidatus Berkiella" species, providing means for identifying members of this order in molecular terms. Twenty four, 7 and 6 CSIs are uniquely shared by members of the genera Legionella, Coxiella and Aquicella, respectively, identifying these groups in molecular terms. The descriptions of these three genera are emended to include information for their novel molecular characteristics. We also describe 12 CSIs that are uniquely shared by D. massiliensis and different members of the genus Rickettsiella. Based on these results, we are proposing an integration of the genus Diplorickettsia with Rickettsiella. Three other CSIs suggest that members of the genera Coxiella and Rickettsiella shared a common ancestor exclusive of other Legionellales. The described molecular markers, due to their exclusivity for the indicated taxa/genera, provide important means for the identification of these clinically important microorganisms and for discovering novel properties unique to them.

A phylogenomic and comparative genomic framework for resolving the polyphyly of the genus Bacillus: Proposal for six new genera of Bacillus species, Peribacillus gen. nov., Cytobacillus gen. nov., Mesobacillus gen. nov., Neobacillus gen. nov., Metabacillus gen. nov. and Alkalihalobacillus gen. nov.

The genus Bacillus, harbouring 293 species/subspecies, constitutes a phylogenetically incoherent group. In the absence of reliable means for grouping known Bacillus species into distinct clades, restricting the placement of new species into this genus has proven difficult. To clarify the evolutionary relationships among Bacillus species, 352 available genome sequences from the family Bacillaceae were used to perform comprehensive phylogenomic and comparative genomic analyses. Four phylogenetic trees were reconstructed based on multiple datasets of proteins including 1172 core Bacillaceae proteins, 87 proteins conserved within the phylum Firmicutes, GyrA-GyrB-RpoB-RpoC proteins, and UvrD-PolA proteins. All trees exhibited nearly identical branching of Bacillus species and consistently displayed six novel monophyletic clades encompassing 5-23 Bacillus species (denoted as the Simplex, Firmus, Jeotgali, Niacini, Fastidiosus and Alcalophilus clades), interspersed with other Bacillaceae species. Species from these clades also generally grouped together in 16S rRNA gene trees. In parallel, our comparative genomic analyses of Bacillus species led to the identification of 36 molecular markers comprising conserved signature indels in protein sequences that are specifically shared by the species from these six observed clades, thus reliably demarcating these clades based on multiple molecular synapomorphies. Based on the strong evidence from multiple lines of investigations supporting the existence of these six distinct 'Bacillus' clades, we propose the transfer of species from these clades into six novel Bacillaceae genera viz. Peribacillus gen. nov., Cytobacillus gen. nov., Mesobacillus gen. nov., Neobacillus gen. nov., Metabacillus gen. nov. and Alkalihalobacillus gen. nov. These results represent an important step towards clarifying the phylogeny/taxonomy of the genus Bacillus.

Robust demarcation of 17 distinct Bacillus species clades, proposed as novel Bacillaceae genera, by phylogenomics and comparative genomic analyses: description of Robertmurraya kyonggiensis sp. nov. and proposal for an emended genus Bacillus limiting it only to the members of the Subtilis and Cereus clades of species.

To clarify the evolutionary relationships and classification of Bacillus species, comprehensive phylogenomic and comparative analyses were performed on >300 Bacillus/Bacillaceae genomes. Multiple genomic-scale phylogenetic trees were initially reconstructed to identify different monophyletic clades of Bacillus species. In parallel, detailed analyses were performed on protein sequences of genomes to identify conserved signature indels (CSIs) that are specific for each of the identified clades. We show that in different reconstructed trees, most of the Bacillus species, in addition to the Subtilis and Cereus clades, consistently formed 17 novel distinct clades. Additionally, some Bacillus species reliably grouped with the genera Alkalicoccus, Caldalkalibacillus, Caldibacillus, Salibacterium and Salisediminibacterium. The distinctness of identified Bacillus species clades is independently strongly supported by 128 identified CSIs which are unique characteristics of these clades, providing reliable means for their demarcation. Based on the strong phylogenetic and molecular evidence, we are proposing that these 17 Bacillus species clades should be recognized as novel genera, with the names Alteribacter gen. nov., Ectobacillus gen. nov., Evansella gen. nov., Ferdinandcohnia gen. nov., Gottfriedia gen. nov., Heyndrickxia gen. nov., Lederbergia gen. nov., Litchfieldia gen. nov., Margalitia gen. nov., Niallia gen. nov., Priestia gen. nov., Robertmurraya gen. nov., Rossellomorea gen. nov., Schinkia gen. nov., Siminovitchia gen. nov., Sutcliffiella gen. nov. and Weizmannia gen. nov. We also propose to transfer 'Bacillus kyonggiensis' to Robertmurraya kyonggiensis sp. nov. (type strain: NB22=JCM 17569T=DSM 26768). Additionally, we report 31 CSIs that are unique characteristics of either the members of the Subtilis clade (containing the type species B. subtilis) or the Cereus clade (containing B. anthracis and B. cereus). As most Bacillus species which are not part of these two clades can now be assigned to other genera, we are proposing an emended description of the genus Bacillus to restrict it to only the members of the Subtilis and Cereus clades.

Comparative genomic analyses of two novel qnrVC6 carrying multidrug-resistant Pseudomonas. spp strains.

OBJECTIVES: This study focused on the comparative genomic analyses of two qnrVC6 carrying Pseudomonas spp. strains which might give us insights on the similarity and difference in the genomic contexts of qnrVC6 gene. METHODS: Comparative genomic analyses of the novel qnrVC6 carrying Pseudomonas spp. genomes with emphasis on their antimicrobial resistance genes and virulence factors were performed. RESULTS: Most Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, Clusters of Orthologous Groups of proteins (COG) categories, and (Gene Ontology) GO terms are shared by both genomes. Although qnrVC6 gene is responsible for the increase of quinolone resistance in both strains, but it duplicated in P. putida strain Guangzhou-Ppu420. And the resistance to ß-lactams and aminoglycosides are dependent on different genes. Sharing some adherence, antiphagocytosis, and iron uptake related genes with P. putida strain Guangzhou-Ppu420, P. aeruginosa strain Guangzhou-Pae617 specifically acquires biosurfactant, pigment, protease, regulation, secretion system, and toxin related virulence factors. CONCLUSIONS: Sharing most KEGG pathways, COG categories, and GO terms, P. putida strain Guangzhou-Ppu420 and P. aeruginosa strain Guangzhou-Pae617 differ in antimicrobial resistance genes and virulence factors.

Whole-genome sequencing and comparative genomic analyses of the medicinal fungus Sanguinoderma infundibulare in Ganodermataceae.

Sanguinoderma infundibulare is a newly discovered species of Ganodermataceae known to have high medicinal and ecological values. In this study, the whole-genome sequencing and comparative genomic analyses were conducted to further understand Ganodermataceae's genomic structural and functional characteristics. Using the Illumina NovaSeq and PacBio Sequel platforms, 88 scaffolds were assembled to obtain a 48.99-Mb high-quality genome of S. infundibulare. A total of 14,146 protein-coding genes were annotated in the whole genome, with 98.6% of complete benchmarking universal single-copy orthologs (BUSCO) scores. Comparative genomic analyses were conducted among S. infundibulare, Sanguinoderma rugosum, Ganoderma lucidum, and Ganoderma sinense to determine their intergeneric differences. The 4 species were found to share 4,011 orthogroups, and 24 specific gene families were detected in the genus Sanguinoderma. The gene families associated with carbohydrate esterase in S. infundibulare were significantly abundant, which was reported to be involved in hemicellulose degradation. One specific gene family in Sanguinoderma was annotated with siroheme synthase, which may be related to the typical characteristics of fresh pore surface changing to blood red when bruised. This study enriched the available genome data for the genus Sanguinoderma, elucidated the differences between Ganoderma and Sanguinoderma, and provided insights into the characteristics of the genome structure and function of S. infundibulare.

Complete genome sequencing and comparative genomic analyses of a new spotted-fever Rickettsia heilongjiangensis strain B8.

Rickettsia heilongjiangensis, a tick-borne obligate intracellular bacterium and causative agent of spotted fever in China, has attracted increasing concern regarding its capability in causing human rickettsiosis. Here, we conducted a genomic analysis of a new R. heilongjiangensis strain B8 (B8) isolated from the serum of a patient who had been bitten by a Haemaphysalis longicornis tick in Anhui Province, China. The present study sought to identify exclusive genes that might be associated with the pathogenicity of B8 using comparative genomics. Specifically, the sequences of B8 were assembled into one circular chromosome of 1,275,081 bp and predicted to contain 1447 genes. Comparative genome analyses were performed based on the genome of B8 and 28 spotted fever group (SFG) rickettsial genomes deposited in NCBI. Phylogenomic analyses indicated the B8 strain was clustered within the R. heilongjiangensis species; however, a sum of 112 and 119 B8-unique genes was identified when compared with R. heilongjiangensis and R. japonica strains, respectively. Functional annotation analyses revealed that these B8-unique genes were mainly annotated to defence mechanisms, lipid transport and metabolism, cell wall/membrane/envelope biogenesis. These data indicate B8 rather represents a previously undescribed human-pathogenic SFG rickettsia lineage, which may be an intermediate lineage of R. heilongjiangensis and R. japonica. Overall, this study isolated a new strain of R. heilongjiangensis in East-Central China for the first time, and provided potential B8-unique genetic loci that could be used for the discrimination of B8 from other R. heilongjiangensis and closely related SFG Rickettsial strains.

Corrigendum: New perspectives on an old grouping: the genomic and phenotypic variability of Oxalobacter formigenes and the implications for calcium oxalate stone prevention.

[This corrects the article DOI: 10.3389/fmicb.2022.1011102.].

Phylogenomics studies and molecular markers reliably demarcate genus Pseudomonas sensu stricto and twelve other Pseudomonadaceae species clades representing novel and emended genera.

Genus Pseudomonas is a large assemblage of diverse microorganisms, not sharing a common evolutionary history. To clarify their evolutionary relationships and classification, we have conducted comprehensive phylogenomic and comparative analyses on 388 Pseudomonadaceae genomes. In phylogenomic trees, Pseudomonas species formed 12 main clusters, apart from the "Aeruginosa clade" containing its type species, P. aeruginosa. In parallel, our detailed analyses on protein sequences from Pseudomonadaceae genomes have identified 98 novel conserved signature indels (CSIs), which are uniquely shared by the species from different observed clades/groups. Six CSIs, which are exclusively shared by species from the "Aeruginosa clade," provide reliable demarcation of this clade corresponding to the genus Pseudomonas sensu stricto in molecular terms. The remaining 92 identified CSIs are specific for nine other Pseudomonas species clades and the genera Azomonas and Azotobacter which branch in between them. The identified CSIs provide strong independent evidence of the genetic cohesiveness of these species clades and offer reliable means for their demarcation/circumscription. Based on the robust phylogenetic and molecular evidence presented here supporting the distinctness of the observed Pseudomonas species clades, we are proposing the transfer of species from the following clades into the indicated novel genera: Alcaligenes clade - Aquipseudomonas gen. nov.; Fluvialis clade - Caenipseudomonas gen. nov.; Linyingensis clade - Geopseudomonas gen. nov.; Oleovorans clade - Ectopseudomonas gen. nov.; Resinovorans clade - Metapseudomonas gen. nov.; Straminea clade - Phytopseudomonas gen. nov.; and Thermotolerans clade - Zestomonas gen. nov. In addition, descriptions of the genera Azomonas, Azotobacter, Chryseomonas, Serpens, and Stutzerimonas are emended to include information for the CSIs specific for them. The results presented here should aid in the development of a more reliable classification scheme for Pseudomonas species.

Development of a Genomics-Based Approach To Identify Putative Hypervirulent Nontyphoidal Salmonella Isolates: Salmonella enterica Serovar Saintpaul as a Model.

While differences in human virulence have been reported across nontyphoidal Salmonella (NTS) serovars and associated subtypes, a rational and scalable approach to identify Salmonella subtypes with differential ability to cause human diseases is not available. Here, we used NTS serovar Saintpaul (S. Saintpaul) as a model to determine if metadata and associated whole-genome sequence (WGS) data in the NCBI Pathogen Detection (PD) database can be used to identify (i) subtypes with differential likelihoods of causing human diseases and (ii) genes and single nucleotide polymorphisms (SNPs) potentially responsible for such differences. S. Saintpaul SNP clusters (n = 211) were assigned different epidemiology types (epi-types) based on statistically significant over- or underrepresentation of human clinical isolates, including human associated (HA; n = 29), non-human associated (NHA; n = 23), and other (n = 159). Comparative genomic analyses identified 384 and 619 genes overrepresented among isolates in 5 HA and 4 NHA SNP clusters most significantly associated with the respective isolation source. These genes included 5 HA-associated virulence genes previously reported to be present on Gifsy-1/Gifsy-2 prophages. Additionally, premature stop codons in 3 and 7 genes were overrepresented among the selected HA and NHA SNP clusters, respectively. Tissue culture experiments with strains representing 4 HA and 3 NHA SNP clusters did not reveal evidence for enhanced invasion or intracellular survival for HA strains. However, the presence of sodCI (encoding a superoxide dismutase), found in 4 HA and 1 NHA SNP clusters, was positively correlated with intracellular survival in macrophage-like cells. Post hoc analyses also suggested a possible difference in intracellular survival among S. Saintpaul lineages. IMPORTANCE Not all Salmonella isolates are equally likely to cause human disease, and Salmonella control strategies may unintentionally focus on serovars and subtypes with high prevalence in source populations but are rarely associated with human clinical illness. We describe a framework leveraging WGS data in the NCBI PD database to identify Salmonella subtypes over- and underrepresented among human clinical cases. While we identified genomic signatures associated with HA/NHA SNP clusters, tissue culture experiments failed to identify consistent phenotypic characteristics indicative of enhanced human virulence of HA strains. Our findings illustrate the challenges of defining hypo- and hypervirulent S. Saintpaul and potential limitations of phenotypic assays when evaluating human virulence, for which in vivo experiments are essential. Identification of sodCI, an HA-associated virulence gene associated with enhanced intracellular survival, however, illustrates the potential of the framework and is consistent with prior work identifying specific genomic features responsible for enhanced or reduced virulence of nontyphoidal Salmonella.

Comparative Genomic Analyses of New and Old World Viscerotropic Leishmanine Parasites: Further Insights into the Origins of Visceral Leishmaniasis Agents.

Visceral leishmaniasis (VL), also known as kala-azar, is an anthropozoonotic disease affecting human populations on five continents. Aetiologic agents belong to the Leishmania (L.) donovani complex. Until the 1990s, three leishmanine parasites comprised this complex: L. (L.) donovani Laveran & Mesnil 1903, L. (L.) infantum Nicolle 1908, and L. (L.) chagasi Lainson & Shaw 1987 (=L. chagasi Cunha & Chagas 1937). The VL causal agent in the New World (NW) was previously identified as L. (L.) chagasi. After the development of molecular characterization, however, comparisons between L. (L.) chagasi and L. (L.) infantum showed high similarity, and L. (L.) chagasi was then regarded as synonymous with L. (L.) infantum. It was, therefore, suggested that L. (L.) chagasi was not native to the NW but had been introduced from the Old World by Iberian colonizers. However, in light of ecological evidence from the NW parasite's enzootic cycle involving a wild phlebotomine vector (Lutzomyia longipalpis) and a wild mammal reservoir (the fox, Cerdocyon thous), we have recently analyzed by molecular clock comparisons of the DNA polymerase alpha subunit gene the whole-genome sequence of L. (L.) infantum chagasi of the most prevalent clinical form, atypical dermal leishmaniasis (ADL), from Honduras (Central America) with that of the same parasite from Brazil (South America), as well as those of L. (L.) donovani (India) and L. (L.) infantum (Europe), which revealed that the Honduran parasite is older ancestry (382,800 ya) than the parasite from Brazil (143,300 ya), L. (L.) donovani (33,776 ya), or L. (L.) infantum (13,000 ya). In the present work, we have now amplified the genomic comparisons among these leishmanine parasites, exploring mainly the variations in the genome for each chromosome, and the number of genomic SNPs for each chromosome. Although the results of this new analysis have confirmed a high genomic similarity (~99%) among these parasites [except L. (L.) donovani], the Honduran parasite revealed a single structural variation on chromosome 17, and the highest frequency of genomic SNPs (more than twice the number seen in the Brazilian one), which together to its extraordinary ancestry (382,800 ya) represent strong evidence that L. (L.) chagasi/L. (L.) infantum chagasi is, in fact, native to the NW, and therefore with valid taxonomic status. Furthermore, the Honduran parasite, the most ancestral viscerotropic leishmanine parasite, showed genomic and clinical taxonomic characteristics compatible with a new Leishmania species causing ADL in Central America.

New perspectives on an old grouping: The genomic and phenotypic variability of Oxalobacter formigenes and the implications for calcium oxalate stone prevention.

Oxalobacter formigenes is a unique bacterium with the ability to metabolize oxalate as a primary carbon source. Most kidney stones in humans are composed of calcium and oxalate. Therefore, supplementation with an oxalate-degrading bacterium may reduce stone burden in patients suffering from recurrent calcium oxalate-based urolithiasis. Strains of O. formigenes are divided into two groups: group I and group II. However, the differences between strains from each group remain unclear and elucidating these distinctions will provide a better understanding of their physiology and potential clinical applications. Here, genomes from multiple O. formigenes strains underwent whole genome sequencing followed by phylogenetic and functional analyses. Genetic differences suggest that the O. formigenes taxon should be divided into an additional three species: Oxalobacter aliiformigenes sp. nov, Oxalobacter paeniformigenes sp. nov, and Oxalobacter paraformigenes sp. nov. Despite the similarities in the oxalyl-CoA gene (oxc), which is essential for oxalate degradation, these strains have multiple unique genetic features that may be potential exploited for clinical use. Further investigation into the growth of these strains in a simulated fecal environment revealed that O. aliiformigenes strains are capable of thriving within the human gut microbiota. O. aliiformigenes may be a better therapeutic candidate than current group I strains (retaining the name O. formigenes), which have been previously tested and shown to be ineffective as an oral supplement to mitigate stone disease. By performing genomic analyses and identifying these novel characteristics, Oxalobacter strains better suited to mitigation of calcium oxalate-based urolithiasis may be identified in the future.

Phylogenomic Analyses and Molecular Signatures Elucidating the Evolutionary Relationships amongst the Chlorobia and Ignavibacteria Species: Robust Demarcation of Two Family-Level Clades within the Order Chlorobiales and Proposal for the Family Chloroherpetonaceae fam. nov.

Evolutionary relationships amongst Chlorobia and Ignavibacteria species/strains were examined using phylogenomic and comparative analyses of genome sequences. In a phylogenomic tree based on 282 conserved proteins, the named Chlorobia species formed a monophyletic clade containing two distinct subclades. One clade, encompassing the genera Chlorobaculum, Chlorobium, Pelodictyon, and Prosthecochloris, corresponds to the family Chlorobiaceae, whereas another clade, harboring Chloroherpeton thalassium, Candidatus Thermochlorobacter aerophilum, Candidatus Thermochlorobacteriaceae bacterium GBChlB, and Chlorobium sp. 445, is now proposed as a new family (Chloroherpetonaceae fam. nov). In parallel, our comparative genomic analyses have identified 47 conserved signature indels (CSIs) in diverse proteins that are exclusively present in members of the class Chlorobia or its two families, providing reliable means for identification. Two known Ignavibacteria species in our phylogenomic tree are found to group within a larger clade containing several Candidatus species and uncultured Chlorobi strains. A CSI in the SecY protein is uniquely shared by the species/strains from this "larger Ignavibacteria clade". Two additional CSIs, which are commonly shared by Chlorobia species and the "larger Ignavibacteria clade", support a specific relationship between these two groups. The newly identified molecular markers provide novel tools for genetic and biochemical studies and identification of these organisms.

Phylogenomics and molecular signatures support division of the order Neisseriales into emended families Neisseriaceae and Chromobacteriaceae and three new families Aquaspirillaceae fam. nov., Chitinibacteraceae fam. nov., and Leeiaceae fam. nov.

The order Neisseriales contains 37 genera harboring 122 species with validly published names, which are placed into two families, Neisseriaceae and Chromobacteriaceae. Genome sequences are now available for 35 of the 37 Neisseriales genera for reliably determining their evolutionary relationships and taxonomy. We report here comprehensive phylogenomic and comparative analyses on protein sequences from 110 Neisseriales genomes plus 3 Chitinimonas genomes using multiple approaches. In a phylogenomic tree based on 596 core proteins, Neisseriales species formed 5 strongly supported clades. In addition to the clades for Neisseriaceae and Chromobacteriaceae families, three novel species clades designated as the "Chitinibacteraceae", "Aquaspirillaceae", and "Leeiaceae" were observed. The genus Chitinimonas grouped reliably with members of the "Chitinibacteraceae" clade. The major clades within the order Neisseriales can also be distinguished based on average amino acid identity analysis. In parallel, our comparative genomic studies have identified 30 conserved signature indels (CSIs) that are specific for members of the order Neisseriales or its five main clades. One of these CSIs is uniquely shared by all Neisseriales, whereas 8, 4, 9, 3 and 5 CSIs are distinctive characteristics of the Neisseriaceae, Chromobacteriaceae, "Chitinibacteraceae", "Aquaspirillaceae" and "Leeiaceae" clades, respectively. Based on the strong phylogenetic and molecular evidence presented here, we are proposing that the three newly identified clades should be recognized as novel families (Chitinibacteraceae fam. nov., Aquaspirillaceae fam. nov. and Leeiaceae fam. nov.) within the order Neisseriales. In addition, we are also emending descriptions of the families Neisseriaceae and Chromobacteriaceae regarding their constituent genera and other distinguishing characteristics.

Major Traditional Probiotics: Comparative Genomic Analyses and Roles in Gut Microbiome of Eight Cohorts.

Modulating gut microbiota to promote host health is well recognized. Therefore, people consume dietary products containing traditional probiotics in wishing to improve their health, and they need more research-based advices on how to select products with suitable probiotic species. Probiotic designers are sometimes confused about how to design precision products for different consumers by taking advantages of different probiotic species' strengths. Additionally, large-scale analyses on traditional probiotic complementarity potentials and their roles in gut microbiome related to common diseases are not well understood. Here, we comprehensively analyzed 444 genomes of major traditional probiotic (sub) species (MTPS, n = 15) by combining one newly sequenced genome with all of the public NCBI-available MTPS-related genomes. The public human fecal metagenomic data (n = 1,815) of eight cohorts were used to evaluate the roles of MTPS, compared to other main gut bacteria, in disease association by examining the species enrichment direction in disease group or the control group. Our work provided a comprehensive genetic landscape and complementarity relations for MTPS and shed light on personalized probiotic supplements for consumers with different health status and the necessity that researchers and manufactures could explore novel probiotics as well as traditional ones.