Haemophilus influenzae is part of the human nasopharyngeal microbiota and a pathogen causing invasive disease. The extensive genetic diversity observed in H. influenzae necessitates discriminatory analytical approaches to evaluate its population structure. This study developed a core genome multilocus sequence typing (cgMLST) scheme for H. influenzae using pangenome analysis tools and validated the cgMLST scheme using datasets consisting of complete reference genomes (N = 14) and high-quality draft H. influenzae genomes (N = 2297). The draft genome dataset was divided into a development dataset (N = 921) and a validation dataset (N = 1376). The development dataset was used to identify potential core genes, and the validation dataset was used to refine the final core gene list to ensure the reliability of the proposed cgMLST scheme. Functional classifications were made for all the resulting core genes. Phylogenetic analyses were performed using both allelic profiles and nucleotide sequence alignments of the core genome to test congruence, as assessed by Spearman\'s correlation and ordinary least square linear regression tests. Preliminary analyses using the development dataset identified 1067 core genes, which were refined to 1037 with the validation dataset. More than 70% of core genes were predicted to encode proteins essential for metabolism or genetic information processing. Phylogenetic and statistical analyses indicated that the core genome allelic profile accurately represented phylogenetic relatedness among the isolates (R 2 = 0.945). We used this cgMLST scheme to define a high-resolution population structure for H. influenzae, which enhances the genomic analysis of this clinically relevant human pathogen.

In the present work, we carried out a comparative genomic analysis to trace the evolutionary trajectory of the bacterial species that make up the Liquorilactobacillus genus, from the identification of genes and speciation/adaptation mechanisms in their unique characteristics to the identification of the pattern grouping these species. We present phylogenetic relationships between Liquorilactobacillus and related taxa such as Bacillus, basal lactobacilli and Ligilactobacillus, highlighting evolutionary divergences and lifestyle transitions across different taxa. The species of this genus share a core genome of 1023 genes, distributed in all COGs, which made it possible to characterize it as Liquorilactobacillus sensu lato: few amino acid auxotrophy, low genes number for resistance to antibiotics and general and specific cellular reprogramming mechanisms for environmental responses. These species were divided into four clades, with diversity being enhanced mainly by the diversity of genes involved in sugar metabolism. Clade 1 presented lower (< 70%) average amino acid identity with the other clades, with exclusive or absent genes, and greater distance in the genome compared to clades 2, 3 and 4. The data pointed to an ancestor of clades 2, 3 and 4 as being the origin of the genus Ligilactobacillus, while the species of clade 1 being closer to the ancestral Bacillus. All these traits indicated that the species of clade 1 could be soon separated in a distinct genus.

BACKGROUND: Aspergillus flavus is an important agricultural and food safety threat due to its production of carcinogenic aflatoxins. It has high level of genetic diversity that is adapted to various environments. Recently, we reported two reference genomes of A. flavus isolates, AF13 (MAT1-2 and highly aflatoxigenic isolate) and NRRL3357 (MAT1-1 and moderate aflatoxin producer). Where, an insertion of 310 kb in AF13 included an aflatoxin producing gene bZIP transcription factor, named atfC. Observations of significant genomic variants between these isolates of contrasting phenotypes prompted an investigation into variation among other agricultural isolates of A. flavus with the goal of discovering novel genes potentially associated with aflatoxin production regulation. Present study was designed with three main objectives: (1) collection of large number of A. flavus isolates from diverse sources including maize plants and field soils; (2) whole genome sequencing of collected isolates and development of a pangenome; and (3) pangenome-wide association study (Pan-GWAS) to identify novel secondary metabolite cluster genes.
RESULTS: Pangenome analysis of 346 A. flavus isolates identified a total of 17,855 unique orthologous gene clusters, with mere 41% (7,315) core genes and 59% (10,540) accessory genes indicating accumulation of high genomic diversity during domestication. 5,994 orthologous gene clusters in accessory genome not annotated in either the A. flavus AF13 or NRRL3357 reference genomes. Pan-genome wide association analysis of the genomic variations identified 391 significant associated pan-genes associated with aflatoxin production. Interestingly, most of the significantly associated pan-genes (94%; 369 associations) belonged to accessory genome indicating that genome expansion has resulted in the incorporation of new genes associated with aflatoxin and other secondary metabolites.
CONCLUSIONS: In summary, this study provides complete pangenome framework for the species of Aspergillus flavus along with associated genes for pathogen survival and aflatoxin production. The large accessory genome indicated large genome diversity in the species A. flavus, however AflaPan is a closed pangenome represents optimum diversity of species A. flavus. Most importantly, the newly identified aflatoxin producing gene clusters will be a new source for seeking aflatoxin mitigation strategies and needs new attention in research.

Campylobacter species are typically helical shaped, Gram-negative, and non-spore-forming bacteria. Species in this genus include established foodborne and animal pathogens as well as emerging pathogens. The accumulation of genomic data from the Campylobacter genus has increased exponentially in recent years, accompanied by the discovery of putative new species. At present, the lack of a standardized species boundary complicates distinguishing established and novel species. We defined the Campylobacter genus core genome (500 loci) using publicly available Campylobacter complete genomes (n = 498) and constructed a core genome phylogeny using 2,193 publicly available Campylobacter genomes to examine inter-species diversity and species boundaries. Utilizing 8,440 Campylobacter genomes representing 33 species and 8 subspecies, we found species delineation based on an average nucleotide identity (ANI) cutoff of 94.2% is consistent with the core genome phylogeny. We identified 60 ANI genomic species that delineated Campylobacter species in concordance with previous comparative genetic studies. All pairwise ANI genomic species pairs had in silico DNA-DNA hybridization scores of less than 70%, supporting their delineation as separate species. We provide the tool Campylobacter Genomic Species typer (CampyGStyper) that assigns ANI genomic species to query genomes based on ANI similarities to medoid genomes from each ANI genomic species with an accuracy of 99.96%. The ANI genomic species definitions proposed here allow consistent species definition in the Campylobacter genus and will facilitate the detection of novel species in the future.IMPORTANCEIn recent years, Campylobacter has gained recognition as the leading cause of bacterial gastroenteritis worldwide, leading to a substantial rise in the collection of genomic data of the Campylobacter genus in public databases. Currently, a standardized Campylobacter species boundary at the genomic level is absent, leading to challenges in detecting emerging pathogens and defining putative novel species within this genus. We used a comprehensive representation of genomes of the Campylobacter genus to construct a core genome phylogenetic tree. Furthermore, we found an average nucleotide identity (ANI) of 94.2% as the optimal cutoff to define the Campylobacter species. Using this cutoff, we identified 60 ANI genomic species which provided a standardized species definition and nomenclature. Importantly, we have developed Campylobacter Genomic Species typer (CampyGStyper), which can robustly and accurately assign these ANI genomic species to Campylobacter genomes, thereby aiding pathogen surveillance and facilitating evolutionary and epidemiological studies of existing and emerging pathogens in the genus Campylobacter.

Streptococcus uberis is a globally endemic and poorly controlled cause of bovine mastitis impacting the sustainability of the modern dairy industry. A core genome was derived from 579 newly sequenced S. uberis isolates, along with 305 publicly available genome sequences of S. uberis isolated from 11 countries around the world and used to develop a core genome multi-locus sequence typing (cgMLST) scheme. The S. uberis core genome comprised 1475 genes, and these were used to identify 1447 curated loci that were indexed into the cgMLST scheme. This was able to type 1012 of 1037 (>97  %) isolates used and differentiated the associated sequences into 932 discrete core genome sequence types (cgSTs). Analysis of the phylogenetic relationships of cgSTs revealed no clear clustering of isolates based on metadata such as disease status or year of isolation. Geographical clustering of cgSTs was limited to identification of a UK-centric clade, but cgSTs from UK isolates were also dispersed with those originating from other geographical regions across the entire phylogenetic topology. The cgMLST scheme offers a new tool for the detailed analysis of this globally important pathogen of dairy cattle. Initial analysis has re-emphasized and exemplified the genetically diverse nature of the global population of this opportunistic pathogen.

A strictly aerobic, Gram-stain-negative, rod-shaped, and motile bacterium, designated strain KMM 296, isolated from the coelomic fluid of the mussel Crenomytilus grayanus, was investigated in detail due to its ability to produce a highly active alkaline phosphatase CmAP of the structural family PhoA. A previous taxonomic study allocated the strain to the species Cobetia marina, a member of the family Halomonadaceae of the class Gammaproteobacteria. However, 16S rRNA gene sequencing showed KMM 296\'s relatedness to Cobetia amphilecti NRIC 0815T. The isolate grew with 0.5-19% NaCl at 4-42 °C and hydrolyzed Tweens 20 and 40 and L-tyrosine. The DNA G+C content was 62.5 mol%. The prevalent fatty acids were C18:1 ω7c, C12:0 3-OH, C18:1 ω7c, C12:0, and C17:0 cyclo. The polar lipid profile was characterized by the presence of phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, and also an unidentified aminolipid, phospholipid, and a few unidentified lipids. The major respiratory quinone was Q-8. According to phylogenomic and chemotaxonomic evidence, and the nearest neighbors, the strain KMM 296 represents a member of the species C. amphilecti. The genome-based analysis of C. amphilecti NRIC 0815T and C. litoralis NRIC 0814T showed their belonging to a single species. In addition, the high similarity between the C. pacifica NRIC 0813T and C. marina LMG 2217T genomes suggests their affiliation to one species. Based on the rules of priority, C. litoralis should be reclassified as a later heterotypic synonym of C. amphilecti, and C. pacifica is a later heterotypic synonym of C. marina. The emended descriptions of the species C. amphilecti and C. marina are also proposed.

Vibrio parahaemolyticus is a significant cause of foodborne illness, and its incidence worldwide is on the rise. It is thus imperative to develop a straightforward and efficient method for typing strains of this pathogen. In this study, we conducted a pangenome analysis of 75 complete genomes of V. parahaemolyticus and identified the core gene mtlA with the highest degree of variation, which distinguished 44 strains and outperformed traditional seven-gene-based MLST when combined with aer, another core gene with high degree of variation. The mtlA gene had higher resolution to type strains with a close relationship compared to the traditional MLST genes in the phylogenetic tree built by core genomes. Strong positive selection was also detected in the gene mtlA (ω > 1), representing adaptive and evolution in response to the environment. Therefore, the panel of gene mtlA and aer may serve as a tool for the typing of V. parahaemolyticus, potentially contributing to the prevention and control of this foodborne disease.

Campylobacteriosis, primarily caused by Campylobacter jejuni and C. coli, is the main bacterial zoonosis worldwide. While poultry is recognized as the main reservoir, bovines are considered another important reservoir for Campylobacter spp. found in human infections. In contrast to chicken, retail beef is seldom contaminated by Campylobacter species. However, beef liver is recognized to be frequently contaminated and has been linked to human infections via epidemiological investigations. Our aims were to evaluate the prevalence of Campylobacter spp. inside and on the surface of beef liver pieces at retail in Finland and to analyse the population in more detail using whole genome sequencing (WGS) to assess the public health relevance. A total of 90 retail beef livers were studied using both enrichment of the external peptone-saline rinse of the liver piece and direct culture from the inside after surface sterilization. Furthermore, 46 of the livers were also studied using direct culture of retail beef liver juice collected from the bottom of the consumer package to estimate the concentration of Campylobacter species. Overall, 44 (49 %) of the samples were positive for Campylobacter species, C. jejuni, C. fetus and C. lari being identified in 42 %, 8.9 % and 1.1 % of the samples, respectively. Direct culture of retail liver juice was a sensitive and convenient method for Campylobacter spp. detection, resulting in 48 % prevalence and a mean concentration of 49 cfu/ml (maximum 335 cfu/ml). Two samples (2.2 %), containing large hepatic ducts, were positive for C. jejuni internally, representing multilocus sequence typing (MLST) sequence type ST-19 and ST-21. WGS, core genome phylogeny and core genome MLST revealed that in most cases only one clearly distinct clone of clinically relevant C. jejuni or C. fetus was isolated from a single lot of samples. However, in some cases several distinct clones were identified simultaneously even from a single liver piece. In epidemiological investigations, it is thus highly advisable to genotype multiple isolates to capture the whole diversity of Campylobacter spp. from suspected food sources. Good kitchen hygiene, avoidance of cross-contamination and thorough cooking are important for limiting the transmission of campylobacteriosis.

As the name of the genus Pantoea (\"of all sorts and sources\") suggests, this genus includes bacteria with a wide range of provenances, including plants, animals, soils, components of the water cycle, and humans. Some members of the genus are pathogenic to plants, and some are suspected to be opportunistic human pathogens; while others are used as microbial pesticides or show promise in biotechnological applications. During its taxonomic history, the genus and its species have seen many revisions. However, evolutionary and comparative genomics studies have started to provide a solid foundation for a more stable taxonomy. To move further toward this goal, we have built a 2,509-gene core genome tree of 437 public genome sequences representing the currently known diversity of the genus Pantoea. Clades were evaluated for being evolutionarily and ecologically significant by determining bootstrap support, gene content differences, and recent recombination events. These results were then integrated with genome metadata, published literature, descriptions of named species with standing in nomenclature, and circumscriptions of yet-unnamed species clusters, 15 of which we assigned names under the nascent SeqCode. Finally, genome-based circumscriptions and descriptions of each species and each significant genetic lineage within species were uploaded to the LINbase Web server so that newly sequenced genomes of isolates belonging to any of these groups could be precisely and accurately identified.

Many Bacillus species are essential antibacterial agents, but their antibiosis potential still needs to be elucidated to its full extent. Here, we isolated a soil bacterium, BP9, which has significant antibiosis activity against fungal and bacterial pathogens. BP9 improved the growth of wheat seedlings via active colonization and demonstrated effective biofilm and swarming activity. BP9 sequenced genome contains 4282 genes with a mean G-C content of 45.94% of the whole genome. A single copy concatenated 802 core genes of 28 genomes, and their calculated average nucleotide identity (ANI) discriminated the strain BP9 from Bacillus licheniformis and classified it as Bacillus paralicheniformis. Furthermore, a comparative pan-genome analysis of 40 B. paralicheniformis strains suggested that the genetic repertoire of BP9 belongs to open-type genome species. A comparative analysis of a pan-genome dataset using the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Cluster of Orthologous Gene groups (COG) revealed the diversity of secondary metabolic pathways, where BP9 distinguishes itself by exhibiting a greater prevalence of loci associated with the metabolism and transportation of organic and inorganic substances, carbohydrate and amino acid for effective inhabitation in diverse environments. The primary secondary metabolites and their genes involved in synthesizing bacillibactin, fencing, bacitracin, and lantibiotics were identified as acquired through a recent Horizontal gene transfer (HGT) event, which contributes to a significant part of the strain`s antimicrobial potential. Finally, we report some genes essential for plant-host interaction identified in BP9, which reduce spore germination and virulence of multiple fungal and bacterial species. The effective colonization, diverse predicted metabolic pathways and secondary metabolites (antibiotics) suggest testing the suitability of strain BP9 as a potential bio-preparation in agricultural fields.