International Journal of Systematic and Evolutionary Microbiology

A strictly anaerobic bacterial strain, F2T, was isolated from an anaerobic bioreactor fermenting source-separated organic waste. Cells of strain F2T are non-spore-forming, rod-shaped (1.5-2.5 x 0.27-0.33 m), and Gram-negative, although they possess a monoderm cell wall architecture. The strain grew at 37 degrees C within a pH range of 5 to 8 and produced short-, branched-, and medium-chain carboxylates as well as ammonium, H2 and CO2, with acetate and propanoate produced or consumed depending on fermentation conditions. The genome consists of a single 2.4 Mbp chromosome with a G+C content of 50.2% and 2,131 predicted genes. Phylogenetic analysis of the 16S rRNA gene against other isolates revealed that strain F2T is most similar to Eubacterium pyruvativorans I-6T (92.06% 16S rRNA identity). Based on further phenotypic, genomic, and phylogenetic analysis, strain F2T represents a novel genus and species within the family Anaerovoracaceae with the proposed name Peptonella octanoica gen. nov. sp. nov. The type strain is F2T (strain accession pending). As a member of this same genus-level clade, we propose reclassifying Eubacterium pyruvativorans as Peptonella pyruvativorans comb. nov. These findings disambiguate Peptonella spp. from the phylogenetically distant and phenotypically distinct Eubacterium limosum ATCC 8486T.

This study investigated the genomic and biochemical characteristics of two amylolytic microbial strains, NATIVEDY160T (= JE7B6T = NRRL B-68523T) and NATIVEDY161T (= JL13D9T, = NRRL B-68524T) isolated from the rumen of healthy Holstein dairy cattle. Both strains are obligately anaerobic, non-motile, Gram positive, catalase-negative, and oxidase-negative. Morphologically, NATIVEDY160T grows in long coccoid chains while NATIVEDY161T grows in short chains or pairs. NATIVEDY160T can catabolize amygdalin, esculin/ferric citrate, and starch, compared to NATIVEDY161T which utilizes amygdalin, arbutin, esculin/ferric citrate, glycogen, and D-maltose as determined by API 50 CH carbon panels. Starch degradation ability was verified for both strains, but neither showed cellulolytic activity as confirmed by starch agar and Congo red agar assays, respectively. HPLC analysis revealed that lactate was the primary end product of both strains carbohydrate fermentation, while strain NATIVEDY161T also produced small amounts of acetate. 16S rRNA sequences from both strains cluster with the Oscillospiraceae (formerly Ruminococcaceae) lineage Ruminococcus species, but average nucleotide identity of either strain compared to closely related Ruminococcus members was under the species threshold (95%). Genomic, phylogenetic, and phenotypic interrogation support NATIVEDY160T and NATIVEDY161T as novel species. Each strain was isolated from the rumen of dairy cows located within the central valley of southern California, which has a rich history of Dutch and Basque dairy farm ownership and is still the case today in the region. In recognition of the contributions and heritage of the central and southern California dairy industry, the names Ruminococcus hollandia and Ruminococcus vasco are proposed with NATIVEDY160T and NATIVEDY161T as their respective type strains.

During mycological surveys in Yunnan Province, China, specimens of a fungus producing massive, upright stromata up to 50 cm high and individually 2.2 Kg in weight were sampled. Through an integrative taxonomic approach combining detailed morphology, multilocus phylogeny (ITS, LSU, RPB2, TUB2), and phylogenomic analyses, this fungus is proposed as the new species Dianjunus rex gen. et sp. nov., the type of the new family Dianjunaceae (Xylariales). Phylogenetic analyses robustly place Dianjunaceae as a distinct sister clade to Graphostromataceae. Divergence time estimation dates the origin of this family to the early Paleocene (~65 Mya), coinciding with the post-K-Pg extinction period, when an estimated 75% of all plant and animal species went extinct, and a significant ecological reorganization of life on earth happened. The stromata of D. rex represent the largest fructifications documented within the Ascomycota, significantly expanding the known morphological range of the Xylariales. The study provides a comprehensive description, including a nodulisporium-like anamorph with periconiella-like branching patterns, and discusses the taxon's phylogenetic placement, and distinctive morphology. This discovery highlights the unexplored fungal diversity in East Asian forests.

Oligella otitidis MSHR-50489EDL strain (ATCC: TSD462; DSMZ: DSM118617) is a new species of the genus Oligella that was isolated from a middle ear discharge swab from a child with chronic suppurative otitis media (CSOM). This Gram-negative coccobacillus produces small, circular, smooth, whitish-opaque and occasionally mucoid colonies. It grows in aerobic conditions at a temperature range from 25-42oC. Phylogenetic analysis demonstrates a relationship to other species of the genera Oligella and average nucleotide identity and digital DNA/DNA hybridization values indicate a distinct species in comparison to other Oligella species. Thus far, the majority of isolates exhibit resistance to ciprofloxacin, the first line treatment for CSOM.

This study describes the characteristics of NATIVEDY162T (= JL13D10T = NRRL B-68525T) a novel bacterium isolated from the rumen of a healthy Holstein dairy cow. NATIVEDY162T was discovered to be an obligately anaerobic, slightly curved, rod that stains Gram-negative, and presents as singlets or short chains. Tests confirmed it is indole-, catalase-, oxidase-negative, and it is not motile. NATIVEDY162T indicated a growth preference within the pH range of 6.5-7.5 with optimal growth at pH 7.0. Carbon panel assays found NATIVEDY162T can utilize D-glucose, L-rhamnose, esculin/ferric citrate, D-lactose, and D-trehalose, whereas weak growth was observed on D-xylose and D-cellobiose. It was also capable of degrading starch and digesting both soluble and insoluble forms of cellulose, with genomic analysis providing further support by revealing a diverse array of carbohydrate-active enzymes (CAZymes) targeting starch and plant structural components like pectin and cellulose. Fermentation of glucose by NATIVEDY162T resulted in the major fermentation products lactate and butyrate. Phylogenetic analysis of the 16S rRNA gene positioned NATIVEDY162T in close relation to other Butyrivibrio species. Genome comparisons using BLAST ANI identified its closest relative as Butyrivibrio proteoclasticus (75.7% identity); however, the similarity did not meet the 95% threshold for species identification. Phylogenetic, genomic, and chemotaxonomic findings highlight differences between NATIVEDY162T and other Butyrivibrio species, indicating it is a novel species. NATIVEDY162T was isolated from a lactating Holstein dairy cow located in Californias San Joaquin Valley, a region rich in Portuguese influence from Azorean migrants who played a key role in the development of the California dairy industry, producing most of the states milk by the mid-20th century. Given this historical context, we propose the name Butyrivibrio azoria type strain NATIVEDY162T in honor of the significant contributions of Azorean and Portuguese dairy farmers in the region.

The genus Erwinia comprises a diverse group of bacteria associated with plants, insects, and the environment, including several economically important phytopathogens. The genus has been revised taxonomically many times, yet a thorough and genome-wide assessment of its evolutionary relationships and genomic diversity has been lacking. In this research, we carried out an extensive phylogenomic and comparative genomic analyses of the genus Erwinia using 104 genomes including historically important strains. Genome-wide analyses integrating average nucleotide identity (ANI), digital DNA-DNA hybridization (dDDH), core-genome phylogenomics, pan-genome analysis, and comparative genomics resolved evolutionary relationships across the genus and identified multiple taxonomic inconsistencies. The pan-genome analysis revealed a relatively small core genome alongside an extensive accessory genome, underscoring the substantial genomic plasticity and ongoing diversification within the genus. The comparative analyses further showed pronounced lineage-specific variation in secretion systems, exopolysaccharide biosynthetic loci, flagellar gene clusters, genomic islands, prophages, and iron acquisition systems, suggesting that virulence-associated determinants have evolved through differential gene gain, loss, and conservation across distinct lineages, thereby facilitating host and ecological niche adaptation. This lineage-specific variation indicates that pathogenicity in the genus is not driven by a single conserved set of virulence determinants but instead reflects distinct combinations of virulence-associated genes. These findings refine the genomic framework of the genus Erwinia, provide evidence for taxonomic revision of several lineages, and improve our understanding of the evolutionary relationships, genomic diversification, and lineage-specific adaptations associated with host interactions and ecological specialization. Impact StatementThis study provides the first comprehensive genome-wide phylogenomic framework for the genus Erwinia, integrating taxonomy, pan-genome diversity, virulence-associated determinants, and mobile genetic elements across all 18 currently recognized species. Analyses resolve evolutionary relationships, uncover multiple taxonomic inconsistencies, identify previously unrecognized species-level lineages, including a putative novel Erwinia species PL328 isolated from Cornus florida (dogwood), and reveal lineage-specific genomic features. These findings establish a valuable genomic foundation for future studies of Erwinia evolution, taxonomy, and plant-microbe interactions. Data SummaryGenomes sequenced in this study were submitted to the NCBI database under the accession numbers: JCBCPT000000000

A facultatively anaerobic, hydrogen-oxidizing, thermophilic bacterium (strain KUI-RBT) was isolated from a geothermal spring biofilm in Rotorua, New Zealand. Strain KUI-RBT is a motile, straight rod, measuring approximately 0.7 {micro}m by 1.0 to 1.5 {micro}m with a diderm cell wall. Growth of KUI-RBT occurred from 39 to 74 {degrees}C (Topt 64.5 {degrees}C), pH 5.0 to 7.5 (pHopt 6.5), and 0 to 1% (w/v) NaCl (NaClopt 0.4-0.7%, w/v). KUI-RBT utilizes carbon dioxide and various organic carbon substrates as carbon sources and hydrogen as an electron donor. KUI-RBT can use oxygen (0-21%, v/v), elemental sulfur, thiosulfate, sulfite, nitrate, arsenate, and selenate as terminal electron acceptors. Major fatty acids of strain KUI-RBT include C20:1, C18:1, and C18:0 and the primary quinone is MTK-7. The whole genome G+C content is 34.23 mol%. Phylogenetic analyses indicate KUI-RBT to be a member of the family Hydrogenothermaceae, with Sulfurihydrogenibium azorense Az-Fu1T its closest characterised relative (94.51% 16S rRNA gene sequence similarity, 78.01% whole genome ANI, 61.34% whole genome AAI). Based on phylogenetic and phenotypic analyses, we propose KUI-RBT represents a novel genus and species within the family Hydrogenothermaceae, for which we propose the name Reysenbachia aerophila gen. nov., sp. nov. The type strain is KUI-RBT (=KCTC accession =JCM accession). The GenBank accession number for the 16S rRNA gene sequence of strain KUI-RBT is PZ052650. The GenBank accession number for the whole genome of strain KUI-RBT is JBVODP000000000.

The obscure life histories of many kickxellalean genera remain a bottleneck for comprehensive taxon sampling and phylogenetic reconstruction of the order. While Kickxellales has long been regarded as saprobes inhabiting soil or dung, the discovery of "amphibious fungi" such as Unguispora, which exhibits dimorphic growth between the animal gut and feces, suggests a cryptic gut-dwelling stage within these genera. Given its phylogenetic proximity to Unguispora, ecophysiological traits of Linderina were investigated to evaluate its potential association with the animal gut. Two isolates of L. macrospora were obtained from Japanese soil, representing the first record of this species in Japan. Physiological assays revealed that the optimal temperature for both vegetative growth and sporulation was 25-30 {degrees}C. Furthermore, comparative growth assays on different media demonstrated that sporocladium abundance per sporangiophore is sensitive to nutrient availability, and nutrient-poor media were determined to be the most suitable for evaluating morphological characterization. Under anaerobic, nutrient-rich conditions which are known to induce yeast-like growth in Unguispora, sporangiospores of L. macrospora produce arthrospores. Although marked morphological plasticity was observed during the arthrospore formation, the occurrence of yeast-like unicellular proliferation suggests a potential relationship with the animal gut. Additionally, vegetative growth and sporulation were markedly inhibited by white light exposure; notably, a lethal effect on growth was observed during incubation at 20 {degrees}C, indicating that the natural niche of the species is restricted to light-shielded environments. Our findings will help to elucidate the cryptic life cycles and evolutionary trajectories within Kickxellales.

Macrolides are key agents for treating infections caused by non-tuberculous mycobacteria (NTM). Nevertheless, chromosomal erm genes conferring inducible macrolide resistance are described in some NTM species, such as Mycobacterium abscessus and M. fortuitum, whereas M. chelonae had long been considered as lacking a functional erm. Recent descriptions from the USA and Japan of a new plasmid-borne erm(55) (erm(55)P) in M. chelonae and other rapidly growing mycobacteria (RGM) have challenged this assumption. We investigated erm(55)P occurrence in clinical RGM referred to the French National Reference Centre for Mycobacteria between 2012 and 2026 by genome screening and erm(55)P specific real-time PCR. Positive isolates underwent long-read whole genome sequencing (GridIon, Oxford Nanopore Technologies). Clarithromycin (CLR) minimum inhibitory concentration (MIC) was determined by broth microdilution (RAPMYCO and FRATMYC, Thermo Fisher) and read up to 14 days. Five clinical isolates showing inducible CLR resistance (MIC range <0.25-64 mg/L on day 3-4 and 128 - >128 mg/L on day 14) were positive for erm(55)P: one M. chelonae, three M. neoaurum, and one M. parafortuitum. erm(55)P-positive M. chelonae genomes from this and previous descriptions did not cluster together in the phylogenetic analysis of 263 genomes. The assembled plasmids showed high similarity to previously reported erm(55)-carrying plasmids, especially within the erm(55)P region. The upstream sequence of erm(55)P showed a secondary structure compatible with a possible translation attenuation mechanism. These findings document the first report of a plasmid-borne erm(55) in Europe in M. chelonae and other RGM and raise concern about the emergence of plasmid macrolide resistance in NTM.

Syngas fermentation by carbon monoxide (CO)-utilising acetogens offers a sustainable route for converting gasified waste materials into value-added chemicals. In this study, we isolated a novel thermophilic CO-utilising bacterium, strain AZ2, from marine hydrothermal sediment collected on the island of Sao Miguel, Azores, Portugal. Strain AZ2 is an obligately anaerobic, spore-forming bacterium. Average nucleotide identity (ANI; 78.4-86.7%) and digital DNA-DNA hybridization (dDDH; 23.4-32.5 %) analyses indicate that strain AZ2 represents a novel species within a previously uncharacterised lineage represented by the GTDB placeholder genus UBA2545 in the Neomoorellaceae family. Strain AZ2 was able to grow fermentatively on CO, producing acetate. We further demonstrated that its closest isolated relatives - Thermanaeromonas toyohensis, T. burensis, and Thermanaeromonas sp. strain 9S - are capable of growing on CO, producing either acetate or hydrogen gas (H2). Additionally, we unveiled the genomic potential for CO utilisation within other members of the GTDB placeholder class DSM-521 (previously Moorellia) to which our isolate belongs, expanding the list of possible thermophilic CO-utilising acetogens. We propose that strain AZ2T represents the type strain of a novel genus and species, named Thermobium azorense gen. nov., sp. nov. (= DSM 121889T = JCM 39698T).

AO_SCPLOWBSTRACTC_SCPLOWUnderstudied bacterial genera present a dynamic phylogenetic landscape and opportunities for discovering new taxa as more strains are isolated and genomic data is added. Here, through phylogenomic analysis, we describe three novel species of the globally distributed cosmopolitan marine bacterial genus Microbulbifer. This genus is ubiquitous in saltwater microbiomes and is a validated source of biodegradation enzymes as well as high value small molecule natural products. Average nucleotide identity (ANI) to the closest known species, Microbulbifer variabilis ATCC 700307T, was less than 88.4% for all three novel species. Isolates of the three novel species, designated as PAAF003T (T = type strain), ZKSA006T, and SSSA003T were imaged to reveal their phormological characteristics. Based on phylogenetic data, strains PAAF003T, ZKSA006T, and SSSA003T represent three new species of the genus Microbulbifer, for which the names Microbulbifer maximicatervae sp. nov., Microbulbifer regidiadema sp. nov., and Microbulbifer mixtoriginis sp. nov. are proposed, respectively, under the SeqCode. We also reconstructed a robust phylogeny of available Microbulbifer genomes, which should faciliatate future isolation and strain description studies.

From Brazilian limestone caves, we isolated 29 bacteria utilizing phenol (23 bacteria), toluene (all bacteria), and/or benzene (all bacteria) as sole carbon sources. One isolate showed phosphate solubilization, while lipase/esterase activity occurred in two isolates; no amylase activity was detected, but 16 isolates ([~]55%) exhibited protease activity. Among them, Nocardioides sp. SF1 was selected for whole-genome sequencing due to its aromatic compound tolerance and protease activity. Additionally, catechol cleavage assays yielded unexpected purple pigmentation, suggesting non-canonical aromatic metabolism. Its high-quality draft genome (4.25 Mbp, 16 contigs, N50 of 887 kb) lacks canonical phenol hydroxylase but encodes alternative oxidation systems, phenylacetyl-CoA pathway, besides, desferrioxamine siderophore, biosurfactants, and phosphate solubilization, key adaptations for oligotrophic caves and biotechnologically interesting activities. Whole-genome comparisons (TYGS/GGDC, OrthoANI and k-mer) suggest potential new species. Lacks acquired antimicrobial resistance genes (ResFinder) and pathogenicity potential (PathogenFinder). Nocardioides sp. SF1 emerges as a non-pathogenic candidate for aromatic bioremediation and plant growth promotion in contaminated, nutrient-poor environments, highlighting cave actinobacterias unexplored biotechnological potential.

The bile acid-converting Peptacetobacter hiranonis is a Gram-positive, anaerobic, potentially spore-forming bacterium. It was first isolated from human feces and was subsequently shown to convert bile acids (BA) in both in vitro and in vivo experiments. The conversion of BA relies on the presence of the 7alpha-dehydroxylation multi-step pathway, encoded by the BA-inducible (bai) operon, harbored by P. hiranonis. In companion animals, P. hiranonis has been characterized as a biomarker for intestinal health, with its loss associated with dysbiosis. However, characterization of P. hiranonis cultured from companion animals is limited. An in-depth characterization of P. hiranonis was published by Chen et al. recently, including the proposal of a new species, Peptacetobacter hominis. We have sequenced the whole genome of both canine- and feline-derived strains of P. hiranonis, characterized these strains biochemically, and assessed their in vitro BA-converting ability as well as their antimicrobial resistance profiles. The strains described here can convert primary into secondary BAs and are whole-genome inhibited by low concentrations of amoxicillin-clavulanate, cefepime, ceftriaxone, chloramphenicol, ciprofloxacin, clindamycin, and metronidazole. Based on whole genome analysis, we propose dividing P. hiranonis into two host-adapted subspecies: P. hiranonis subsp. deconjugans and P. hiranonis subsp. nondeconjugans, based on their genomic differences and divergent ability to deconjugate BAs; a function that appears widely distributed among P. hiranonis strains cultured from dogs, but absent from those cultured from cats. Taken together, our results confirmed the BA conversion ability of P. hiranonis cultured from dogs and cats and reveal host-associated genomic and functional differences within the species.

Endosymbiosis is widespread throughout the tree of life. Understanding how the transition of a bacterial endosymbiont from facultative to host-dependent obligate life occurs is an important question for defining the origin of endosymbiosis. A novel gram-positive bacillus, Brevibacillus sp. TJ4 was isolated from the nitrogen-fixing yeast Rhodotorula mucilaginosa JGTA-S1, which houses several endobacteria within its cells. TJ4 can survive independently of yeast but exhibits genomic and metabolic features characteristic of an evolving endosymbiont, slowly assuming a host-dependent, obligate lifestyle. The TJ4 genome contains several incomplete pathways for carbohydrate, amino acid, vitamin, and cofactor metabolism, which is reflected in its increased reliance on host-derived nutrients and auxotrophy compared with that of other Brevibacillus spp. Comparative genomics revealed widespread genome rearrangements, loss of synteny, and multiple cross-genus and inter-kingdom horizontal gene transfer (HGT) events in TJ4 compared to other Brevibacillus spp. These HGTs include the acquisition of genes from bacteriophages and co-resident endobacteria of JGTA-S1. One such horizontally acquired gene, Type II 3-dehydroquinate dehydratase (AroQ), appears to have originated from the Rhodotorula host itself. This acquisition functionally restores the shikimate pathway in strain TJ4, as evidenced by the phylogenetic placement of AroQ from TJ4 within the clade of fungal AroQ homologs. Potential exploitation of the host JGTA-S1 appears to be a probable mode of endosymbiosis of TJ4, an evolving endosymbiont that we named Brevibacillus rhodotorulae sp. nov.

Knowledge of the family Lactobacillaceae rests largely on isolates from foods and a few repeatedly sampled hosts. Reference databases give an unrecognised sequence the name of its nearest relative, so a lineage with no entry of its own is renamed rather than flagged. Here we classify the family across the public plant and invertebrate amplicon record to locate the hosts carrying undescribed lineages. Across 3,344 independent 16S rRNA gene amplicon studies, every sequence cluster was tested against a type-strain reference and placed at the deepest rank it supports. Of 204,813 classified clusters, 20,516 were named to species and 127,906 to genus, while 4,741 matched no described species. Six published datasets whose authors could name their Lactobacillaceae only as "Lactobacillus", or not at all, are reclassified here. Genera described from one habitat occur far beyond it, three bee-associated genera occurring on Rosaceae and Brassicaceae at several times their rate on wind-pollinated grasses, and we found no published Bombilactobacillus record from a plant. Undescribed lineages concentrate in the least-cultured hosts, reaching 86.1% and 77.4% of studies in the stingless bees Melipona and Tetragonula, and are most divergent in the turtle ant Cephalotes and, among plants, in tea, Camellia. An independent genome-resolved survey of pot honey converges on the same two genera. The primary descriptions of forty-eight species from these hosts specify a supplemented medium, so the hosts carrying undescribed lineages also indicate how to culture them.

The cyanobacterium Dolichospermum circinale is a known producer of the neurotoxin saxitoxin and its analogues, collectively known as the paralytic shellfish toxins (PSTs). PSTs vary in potency, and the reported toxin profiles of D. circinale blooms vary in the quantities of individual PSTs, with the regulation of these profiles being poorly understood. In this study, we present the genomes of four D. circinale strains (ACBU01, ACMB03, ACMB13 and FSS-124) with unique PST profiles and perform genome-wide comparisons and specific analysis of the PST-producing biosynthetic gene cluster (sxt) to understand the variability in PST quotas. A reassessment of the previously published D. circinale AWQC131C genome was also performed to collate genomic variation between all strains. Analysis at the nucleotide and amino acid sequence level revealed that toxic strains maintain high genome-wide similarities, corroborated by the analysis of the pan- and variable genomes of each strain. Specifically, the sxt gene sequences were 99-100% identical across all strains. Novel tailoring (sxtSUL, sxtDIOX) and transport (sxtM4) genes were identified within the sxt cluster that were not reported previously in D. circinale. Taken together, these results indicate that the genetic machinery involved in PST production is conserved in this species, suggesting that the regulation of PST biosynthesis in D. circinale does not occur at the genomic level.

Plant growth-promoting bacterial endophytes represent a sustainable strategy for enhancing agricultural productivity while reducing reliance on synthetic fertilizers and pesticides. This study focused on the genomic and functional characterization of two endophytic bacterial strains, R11F and R19M, isolated from bean and maize roots, respectively. Comparative analyses based on 16S rRNA gene sequences, average nucleotide identity (ANI), and genome-to-genome distance calculations (GGDC) classified both isolates as Pseudomonas palleroniana. Comparative genomic analyses revealed highly conserved genomes containing genes associated with plant colonization, phosphate solubilization, stress adaptation, heavy metal resistance, and hydrocarbon degradation. Genome mining further identified 17 and 18 biosynthetic gene clusters (BGCs) in R11F and R19M, respectively, including non-ribosomal peptide synthetases (NRPS), pyoverdine, NRP-metallophores, RiPP-like compounds, arylpolyenes, {beta}-lactones, terpenes, NAGGN, and hydrogen cyanide. Strain-specific BGCs associated with syringomycin and viscosin biosynthesis were identified in R11F, whereas R19M harbored clusters related to asplenin and kolossin biosynthesis. In vitro assays confirmed indole production, phosphate solubilization, and siderophore production, as well as the ability of both strains to grow in nitrogen-free medium. Both strains significantly inhibited the growth of Fusarium oxysporum, Phytophthora cinnamomi, and Colletotrichum gloeosporioides. Furthermore, plant inoculation assays demonstrated host-dependent growth promotion, with R11F showing the most consistent improvements in plant growth parameters in tomato, wheat, and lentil. Overall, the integration of comparative genomics and experimental validation demonstrates that P. palleroniana R11F and R19M possess complementary traits associated with plant growth promotion, pathogen suppression, saline stress adaptation, and bioremediation.

Multilocus sequence typing (MLST) remains the predominant method for typing bacterial strains. A common method for investigating particularly successful epidemic lineages within a species is to cluster isolates with similar MLST profiles into clonal complexes (CCs). Some CCs, such as international clones (ICs) in A. baumannii, are identified with specific sequence types (STs) and are of particular importance to human health. Although theoretically simple, there is a lack of convenient, user-friendly tools perform this analysis. Here we present PhyloMLST, a tool to cluster bacterial isolates into CCs and map them to ICs using the output from existing MLST tools and user-provided STs. As there is potential for aberrant IC assignment arising from excessively large CCs constructed with spurious links, PhyloMLST provides additional functionality to correct IC assignment with a user-provided phylogenetic tree. Although designed with A. baumannii in mind, PhyloMLST can be applied to any bacteria where construction and investigation of CCs based on MLST is performed. Impact statementMany bacterial pathogens are characterised by successful epidemic lineages that are responsible for a substantial number of infections, may be more virulent, and may carry an abundance of antimicrobial resistance genes. These epidemic lineages are comprised of a number of multilocus sequence typing (MLST) sequence types (STs), clustered into clonal complexes (CCs). To date, identifying which STs belong to which epidemic lineage has been challenging, with no simple analytical tools available. Here, we present PhyloMLST - a phylogenetically-aware method for assigning STs to epidemic lineages. The customisable nature of the tool will enable researchers to straightforwardly characterise any population of bacteria that they are working on using MLST data and user-defined definitions of epidemic lineages. Data summaryThe PhyloMLST source code and example data shown here is available at https://github.com/Mattn286/PhyloMLST.

Xenorhabdus bovienii is a symbiotic bacterium associated with entomopathogenic nematodes of the genus Steinernema. Comparative genomic analyses of closely related isolates provide an opportunity to investigate fine-scale diversification, genome plasticity, and the evolutionary processes shaping symbiotic bacterial populations. Here, we analyzed four X. bovienii isolates (XenUTI4.1-XenUTI4.4) recovered from a single Steinernema feltiae isolation event using comparative genomics approaches integrating average nucleotide identity (ANI), single-nucleotide polymorphism (SNP) analyses, pangenome reconstruction, biosynthetic gene cluster (BGC) prediction, and mobile element-associated annotation screening. Whole-genome comparisons revealed extremely high genomic similarity among isolates, with ANI values exceeding 99.84%. Read-based SNP analyses identified only 23-36 annotated variants relative to the XenUTI4.1 reference genome, indicating limited sequence divergence despite detectable microvariation. Functional annotation of these variants showed that most corresponded to missense or synonymous substitutions affecting a small number of coding sequences. Pangenome analysis identified 4,712 orthologous gene clusters, including a highly conserved core genome of 4,256 clusters (90.3%) shared by all isolates and a relatively small accessory genome comprising 456 clusters. antiSMASH analyses revealed broadly conserved secondary metabolite biosynthetic potential across the four genomes, whereas screening of genome annotations identified abundant phage-related, transposase-associated, and recombination-associated genes consistent with ongoing genome plasticity. Collectively, these results demonstrate that the analyzed X. bovienii isolates represent a highly conserved population exhibiting limited but detectable genomic microdiversification. The coexistence of a large core genome, a modest accessory gene complement, and numerous mobile element-associated functions suggests that localized sequence variation and mobile genetic elements contribute to genomic diversification within S. feltiae-associated X. bovienii populations.

Bartonella henselae is a zoonotic pathogen associated with cat-scratch disease. Although multilocus sequence typing (MLST) has been used for strain classification, its resolution for distinguishing between B. henselae isolates remains limited. We herein developed a B. henselae-specific core genome MLST (cgMLST) scheme based on whole-genome sequencing data and examined the genetic and phenotypic diversities of 80 strains derived from cats, humans, mongooses, and masked palm civets. Using the conventional MLST scheme, the 80 strains were classified into nine sequence types (STs), while cgMLST subdivided them into 72 cgSTs, demonstrating a marked improvement in discriminatory power. The cgMLST scheme comprised 1,183 core genes and showed high applicability across the 80 strains. A phylogenetic analysis revealed that ST1, which has been associated with cat-scratch disease, was further subdivided into three major clusters and two singletons, indicating high genetic heterogeneity within this ST. We also found that the bafA subtypes clustered in a manner that was largely consistent with the cgMLST-based phylogenetic structure, suggesting a close relationship between bafA variations and the genomic background of B. henselae strains. In a human umbilical vein endothelial cell proliferation assay, strains belonging to distinct cgSTs exhibited strain-dependent differences in proliferative capacity, which were associated with the bafA subtype classification. Some strains induced focal cell fragmentation and a reduced cell density at a high multiplicity of infection, indicating strain-dependent differences in endothelial cell injury. Collectively, the present results establish a high-resolution cgMLST framework for B. henselae and demonstrate that genetically distinct strains have diverse endothelial cell phenotypes.