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The constant battle between bacteria and viruses has led to the development of sophisticated antiviral defense strategies by bacteria to defend themselves against phages. This study analyzed a marshland metagenome to identify and characterize bacterial antiviral defense systems and phage interactions. We assembled 210 metagenome-assembled genomes (MAGs) from environmental DNA extracted from Pallikaranai marshland soil and 37 unclassified MAGs were filtered. MIMAG standards were followed, 2 high-quality and 15 medium-quality unclassified MAGs were picked. MINCED was used to identify 137 CRISPR arrays in the quality MAGs, and ViroBLAST was used to identify the phages that interact with the bacteria. About 242 spacer sequences were extracted from the CRISPR arrays, of which 54 had significant matches in the ViroBLAST database. 7 unverified bacteriophage species were also detected in the MAGs. The viral group of Caudoviricetes phage elements were identified as a frequent genome terminal repeat. The PADLOC identified 11 genes involved as a defense system in the MAGs. The PD-T4-6 defense system was found to be prevalent in 15 different unclassified MAGs. This study presents valuable insights intothe adaptations of unclassified bacteria to bacteriophages, as well as the genes used by these bacteria as a defense mechanism., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The gut microbiota is a treasure trove of carbohydrate-active enzymes (CAZymes). To explore novel and efficient CAZymes, we analyzed the 4,142 metagenome-assembled genomes (MAGs) of the horse gut microbiota and found the MAG117.bin13 genome (Bacteroides fragilis) contains the highest number of polysaccharide utilisation loci sites (PULs), indicating its high capability for carbohydrate degradation. Bioinformatics analysis indicate that the PULs region of the MAG117.bin13 genome encodes many hypothetical proteins, which are important sources for exploring novel CAZymes. Interestingly, we discovered a hypothetical protein (595 amino acids). This protein exhibits potential CAZymes activity and has a lower similarity to CAZymes, we named it BfLac2275. We purified the protein using prokaryotic expression technology and studied its enzymatic function. The hydrolysis experiment of the polysaccharide substrate showed that the BfLac2275 protein has the ability to degrade α-lactose (156.94 U/mg), maltose (92.59 U/mg), raffinose (86.81 U/mg), and hyaluronic acid (5.71 U/mg). The enzyme activity is optimal at pH 5.0 and 30 ℃, indicating that the hypothetical protein BfLac2275 is a novel and multifunctional CAZymes in the glycoside hydrolases (GHs). These properties indicate that BfLac2275 has broad application prospects in many fields such as plant polysaccharide decomposition, food industry, animal feed additives and enzyme preparations. This study not only serves as a reference for exploring novel CAZymes encoded by gut microbiota but also provides an example for further studying the functional annotation of hypothetical genes in metagenomic assembly genomes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Phenolic acids (PAs) are widely distributed allelochemicals in various environments. To better understand the fate of PAs in environments, a halotolerant PAs-degrading bacterium (named strain RR2S18 T ) isolated from rhizosphere soil was identified as a novel species of Devosia, named Devosia rhizosphaerae sp. nov. The strain initially degraded PAs into central ring-fission intermediates (protocatechuic acid) using the CoA-dependent non-β-oxidation pathway. The produced ring-fission intermediates were then consecutively degraded by an ortho-cleavage reaction and the β-ketoadipic acid pathway. A comparative genomics analysis of 62 Devosia strains revealed that PAs-degrading genes were ubiquitous in their genomes, indicating that PAs degradation is universal among members of this genus. The analysis also suggested that the genes involved in CoA-dependent non-β-oxidation are inherent to Devosia strains, while those involved in ring-fission and β-ketoadipic acid pathways were obtained by horizontal gene transfer., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Bacillus species are extensively documented as plant growth-promoting rhizobacteria, contributing significantly to the enhancement of soil fertility, nutrient recycling, and the control of phytopathogens. Utilizing them as biocontrol agents represents an environmentally friendly strategy, particularly within the rhizospheric community. This study presents the comprehensive genome sequences of three B. velezensis strains (LGMB12, LGMB319, and LGMB426) which were previously isolated from root samples of maize (Zea mays L.), along with a type strain FZB42. The research assesses the capability of the three strains for antagonizing fungi, specifically Fusarium graminearum, Fusarium verticillioides, Colletotrichum graminicola, and Stenocarpella sp. In paired cultures involving maize fungi, treatments containing bacteria B. velezensis exhibited statistically significant differences compared to both negative and positive treatments in terms of antagonism. Furthermore, genome mining techniques were employed to explore their inherent antagonistic potential. The assembly revealed that strains LGMB12, LGMB319, LGMB426, and FZB42 exhibit genome sizes of 4,187,541 bp, 4,244,954 bp, 3,976,537 bp, and 3,990,518 respectively. Their respective G + C content stands at 46.42 %, 46.50 %, 46.51 %, and 46.38 %. Moreover, the genomes present multiple gene clusters responsible for the synthesis of secondary metabolites and carbohydrate-active enzymes (CAZymes). These clusters highlight a diverse array of antibacterial and antifungal properties, complemented by numerous plant growth-promoting genes. These results highlight the potential of B. velezensis LGMB12, LGMB319, and LGMB426 strains as biocontrol and plant growth promotion agents, being promising candidates for further studies in agricultural production, including field trials., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Evidence has indicated that Enterococcus plays a vital role in non-alcoholic fatty liver disease (NAFLD) development. However, the microbial genetic basis and metabolic potential in the disease are yet unknown. We previously isolated a bacteria Enterococcus faecium B6 (E. faecium B6) from children with NAFLD for the first time. Here, we aim to systematically investigate the potential of strain B6 in lipogenic effects. The lipogenic effects of strain B6 were explored in vitro and in vivo. The genomic and functional characterizations were investigated by whole-genome sequencing and comparative genomic analysis. Moreover, the metabolite profiles were unraveled by an untargeted metabolomic analysis. We demonstrated that strain B6 could effectively induce lipogenic effects in the liver of mice. Strain B6 contained a circular chromosome and two circular plasmids and posed various functions. Compared to the other two probiotic strains of E. faecium, strain B6 exhibited unique functions in pathways of ABC transporters, phosphotransferase system, and amino sugar and nucleotide sugar metabolism. Moreover, strain B6 produced several metabolites, mainly enriched in the protein digestion and absorption pathway. The unique potential of strain B6 in lipogenic effects was probably associated with glycolysis, fatty acid synthesis, and glutamine and choline transport. This study pioneeringly revealed the metabolic characteristics and specific detrimental traits of strain B6. The findings provided new insights into the underlying mechanisms of E. faecium in lipogenic effects, and laid essential foundations for further understanding of E. faecium-related disease., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Vibrio spp., known as significant marine pathogens, have become more prevalent due to global warming. Antibiotics released into the environment drive Vibrio resistance. The increasing consumption of seafood leads to more interactions between Vibrio and humans. Despite this concerning trend, there remains a lack of large-scale surveillance for Vibrio contamination across various types of food. This study isolated 4027 Vibrio strains, primarily comprising V. parahaemolyticus and V. alginolyticus, in 3581 fresh shrimp and meat products from 2013 to 2022. The Vibrio strains showed increased resistance to important antibiotics, especially β-lactams used to treat foodborne bacterial infections. Whole genome sequencing of 591 randomly chosen strains showed a strong correlation between antibiotic resistance and genotypes in Vibrio. Notably, various ESBL genes have evolved over the past 8 years, with bla VEBs being the most dominant. Additionally, carbapenemase genes, such as bla NDM-1 , have become increasingly prevalent in recent years. Various mobile genetic elements, including IncQ and IncA/C plasmids, recoverable in Vibrio, facilitate the transmission of crucial β-lactamase genes. These data provide insights into the evolutionary traits of antimicrobial resistance in foodborne Vibrio strains over a decade. Policymakers should consider these findings when devising appropriate strategies to combat bacterial antimicrobial resistance and safeguard human health., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Few aerobic hyperthermophilic microorganisms degrade polysaccharides. Here, we describe the genome-enabled enrichment and optical tweezer-based isolation of an aerobic polysaccharide-degrading hyperthermophile, Fervidibacter sacchari, previously ascribed to candidate phylum Fervidibacteria. F. sacchari uses polysaccharides and monosaccharides for growth at 65-87.5 °C and expresses 191 carbohydrate-active enzymes (CAZymes) according to RNA-Seq and proteomics, including 31 with unusual glycoside hydrolase domains (GH109, GH177, GH179). Fluorescence in-situ hybridization and nanoscale secondary ion mass spectrometry confirmed rapid assimilation of 13 C-starch in spring sediments. Purified GHs were optimally active at 80-100 °C on ten different polysaccharides. Finally, we propose reassigning Fervidibacteria as a class within phylum Armatimonadota, along with 18 other species, and show that a high number and diversity of CAZymes is a hallmark of the phylum, in both aerobic and anaerobic lineages. Our study establishes Fervidibacteria as hyperthermophilic polysaccharide degraders in terrestrial geothermal springs and suggests a broad role for Armatimonadota in polysaccharide catabolism., (© 2024. The Author(s).)

This study aimed to evaluate the safety profile and beneficial effects of Heyndrickxia coagulans strain BC99 (BC99) for potential use in functional foods and pharmaceuticals. We began with whole genome sequencing of BC99, followed by a comprehensive safety assessment comprising genome analysis, hemolysis, cytotoxicity, antibiotic susceptibility tests, and cell adhesion and tolerance studies, along with acute and subacute oral toxicity studies in animal models. BC99 was isolated from a well-characterized collection originating from the feces of a healthy infant. Our results indicated no hemolytic activity on Columbia blood agar plates and broad antibiotic sensitivity, including to gentamicin, ampicillin, chloramphenicol, ciprofloxacin, and others. Cytotoxicity testing confirmed no adverse effects on HT-29 cells and significant adhesive properties to intestinal epithelial cells. Tolerance tests demonstrated over 90% viability of BC99 under simulated gastrointestinal conditions. In vivo studies in mice and rats confirmed the absence of adverse effects following oral administration. Collectively, these findings support BC99's robust tolerance to gastrointestinal environments, strong adhesion capabilities, and a broad spectrum of antibiotic resistance, underlining its potential as a safe and effective agent for gut microbiota modulation and host health enhancement., (© 2024. The Author(s).)

Objectives: To investigate the characteristics and clonal dynamics of tigecycline-resistant Acinetobacter baumannii (TRAB) isolates from a Chinese hospital from 2016 to 2021., Methods: A total of 64 TRAB isolates were screened and WGS was performed. Phylogenetic analysis and non-polymorphic mutation analysis were used to analyse their clonal dynamics and tigecycline resistance-related mutations. RT-PCR was used to analyse the expression of the resistance-nodulation cell-division (RND) efflux pump genes adeB and adeJ. Gene cloning was used to explore the effect of tet(39) variants on tigecycline resistance., Results: Most TRAB isolates were found to be MDR, with 95% (61/64) of the isolates showing resistance to carbapenems. These TRAB isolates were classified into three primary genetic clusters based on core-genome SNPs. The KL2 cluster persisted throughout the study period, whereas the KL7 cluster emerged in 2019 and became the dominant clone. The KL7 cluster carried more antimicrobial resistance genes than the other two clusters. The predominant tigecycline resistance mechanism of the KL2 cluster and KL7 cluster was IS insertion in adeN (82.1%, 23/28) and genetic alterations in adeS (76.2%, 16/21), respectively. Eleven novel AdeS mutations were identified associated with elevated AdeB expression and tigecycline resistance. Moreover, we characterized a plasmid-borne tet(39) variant with an Ala-36-Thr substitution that synergizes with the RND efflux pump to confer high-level tigecycline resistance., Conclusions: This work provides important insights into the diverse mechanisms associated with tigecycline resistance in A. baumannii, highlighting a pressing need for further monitoring of ST2-KL7 A. baumannii in clinical settings., (© The Author(s) 2024. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

A Gram-stain-negative bacterium, designated strain LY-5  T , was isolated from an activated sludge sample collected from a river in Yancheng city, Jiangsu province, China. Cells of strain LY-5  T , were strictly aerobic, non-motile and rod-shaped. Growth occurred at 15-37 °C (optimum, 30 °C), at pH 5.0-9.0 (optimum 7.0) and at 0-3% (w/v) NaCl (optimum, 0%). Phylogenetic analysis based on 16S rRNA gene and genome sequences indicated that strain LY-5  T formed a distinct phyletic branch within the family Chitinophagaceae, with closest relatives being members of the genera Phnomibacter, Aurantisolimonas, and Chitinophaga, sharing 88.5-90.3% sequence similarity. Moreover, the average amino acid identity (AAI) between strain LY-5  T and its closed phylogenetic neighbors was below 70%, indicating it belongs to a novel genus. The predominant cellular fatty acids of LY-5  T were iso-C 15:0 , iso-C 15:1 G and summed feature 3 (C 16:1 ω7c and/or C 16:1 ω6c), and the only isoprenoid quinone was menaquinone-7 (MK-7). The major polar lipids identified in strain LY-5  T comprised phosphatidylethanolamine, two unidentified aminolipids and five unidentified lipids. The genome size of strain LY-5  T was 4.8 Mbp with a G + C content of 38.9%. Based on the evidence presented in this study, strain LY-5  T represents a novel species of a new genus in the family Chitinophagaceae, for which the name Polluticaenibacter yanchengensis gen. nov., sp. nov. (Type strain LY-5  T  = CCTCC AB 2023260  T  = KCTC 102218  T ) is proposed., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Listeria monocytogenes (LM) is a zoonotic pathogen that causes sporadic infectious listeriosis, which is a foodborne disease associated with consumption of contaminated food or feed. The internal organs of an African lion from a zoo in Shanghai were analysed to determine the cause of death. LM infection was suspected on the basis of the clinical symptoms and pathological changes and confirmed by polymerase chain reaction, whole genome sequencing and phylogenetic analysis. This is the first report of LM infection of an African lion in China., (© 2024 The Author(s). Veterinary Medicine and Science published by John Wiley & Sons Ltd.)

Vulnerable patients in an intensive care unit (ICU) setting are at high risk of infection from bacteria including gut-colonising Escherichia coli and Klebsiella species. Complex ICU procedures often depend on successful antimicrobial treatment, underscoring the importance of understanding the extent of patient colonisation by multi-drug-resistant organisms (MDROs) in large UK ICUs. Previous work on ICUs globally uncovered high rates of colonisation by transmission of MDROs, but the situation in UK ICUs is less understood. Here, we investigated the diversity and antibiotic resistance gene (ARG) carriage of bacteria present in one of the largest UK ICUs at the Queen Elizabeth Hospital Birmingham (QEHB), focusing primarily on E. coli as both a widespread commensal and a globally disseminated multi-drug-resistant pathogen. Samples were taken during highly restrictive coronavirus disease 2019 (COVID-19) control measures from May to December 2021. Whole-genome and metagenomic sequencing were used to detect and report strain-level colonisation of patients, focusing on E. coli sequence types (STs), their colonisation dynamics and antimicrobial resistance gene carriage. We found a lack of multi-drug resistance (MDR) in the QEHB. Only one carbapenemase-producing organism was isolated, a Citrobacter carrying bla KPC-2 . There was no evidence supporting the spread of this strain, and there was little evidence overall of nosocomial acquisition or circulation of colonising E. coli . Whilst 22 different E. coli STs were identified, only 1 strain of the pandemic ST131 lineage was isolated. This ST131 strain was non-MDR and was found to be a clade A strain, associated with low levels of antibiotic resistance. Overall, the QEHB ICU had very low levels of pandemic or MDR strains, a result that may be influenced in part by the strict COVID-19 control measures in place at the time. Employing some of these infection prevention and control measures where reasonable in all ICUs might therefore assist in maintaining low levels of nosocomial MDR.

Background: This study aimed to assess the prevalence and genomic characteristics of Shiga-toxigenic (STEC) and Enteroaggregative E. coli (EAEC) strains in raw mussels and ready-to-eat (RTE)-stuffed mussels, focusing on potential public health implications for identifying virulence and antimicrobial resistance genes., Results: The genome sequence analysis identified the E. coli strain named 23EM as serotype O111:H12, with adhesion (fimH-54) and fumarate hydratase (fumC-11) genes. The draft genome (4.9 Mb, 50.6% GC content, 111 contigs, 4,688 genes) is available in NCBI GenBank (accession JAWXVJ000000000). The strain, classified as ST292 and CC ST10, showed high similarity to nonpathogenic E. coli MG1655 but was distinct from pathogenic strains such as EAEC and ExPEC. In silico serotyping revealed the presence of O111-antigen flippase (wzx) and H12-antigen flagellin (fliC) genes. The strain harbors an IncFII (pCoo) plasmid with 96.95% identity. PathogenFinder predicted a 92% probability of being a human pathogen, supported by 720 pathogenic protein families. CRISPR analysis identified one high-evidence sequence with nine spacers and six low-evidence sequences. Phylogenetic analysis using RAxML positioned 23EM close to nonpathogenic E. coli but distant from other pathogenic strains. Antimicrobial resistance genes across multiple classes, including macrolides, fluoroquinolones, and aminoglycosides, were identified. The strain also contains several virulence factors, such as adhesins (e.g., ECP, ELF, TIF, type IV pili), and autotransporter genes (espP, pic), highlighting its significant pathogenic potential and public health risk., Conclusions: This study highlights the ability of the detection of E. coli strains harboring virulence and antimicrobial resistance genes in mussels, thus emphasizing the importance of ongoing surveillance and careful consideration of the potential risks associated with the consumption of these shellfish., (© 2024. The Author(s).)

Background: Zoonotic human tuberculosis (TB) caused by Mycobacterium bovis (M. bovis) is as vital as Mycobacterium tuberculosis, however with scarce available information. We aimed to use whole-genome sequencing (WGS) technology to take a deep insight into the circulating genotypes of human M. bovis and the genomic characteristics underlying virulence and drug resistance., Methods: The study included smear positive Ziehl-Neelsen samples from patients with suspected tuberculosis. Samples were cultured on Lowenstein-Jensen media and suspected colonies of M. bovis were selected to undergo DNA extraction and WGS. Data was analysed using the Bacterial and Viral Bioinformatics Resource Center (BV-BRC), and online bioinformatics tools. A phylogenetic tree was constructed for our sequenced strains, in addition to a set of 59 previously sequenced M. bovis genomes from different hosts and countries., Results: Out of total 112 mycobacterial positive cultures, five M. bovis were isolated and underwent WGS. All sequenced strains belonged to Mycobacterium tuberculosis var bovis, spoligotype BOV_1; BOV_11. Resistance gene mutations were determined in 100% of strains to pyrazinamide (pncA and rpsA), isoniazid (KatG and ahpC), ethambutol (embB, embC, embR and ubiA), streptomycin (rpsl) and fluoroquinolones (gyrA and gyrB). Rifampin (rpoB and rpoC) and delamanid (fbiC) resistance genes were found in 80% of strains. The major represented virulence classes were the secretion system, cell surface components and regulation system. The phylogenetic analysis revealed close genetic relatedness of three sequenced M. bovis strains to previous reported cow strains from Egypt and human strains from France, as well as relatedness of one M. bovis strain to four human Algerian strains. One sequenced strain was related to one cow strain from Egypt and a human strain from South Africa., Conclusions: All sequenced M. bovis isolates showed the same spoligotype, but diverse phylogeny. Resistance gene mutations were detected for anti-TB drugs including pyrazinamide, isoniazid, streptomycin, ethambutol, fluoroquinolones, cycloserine, rifampin and delamanid. The virulence profile comprised genes assigned mainly to secretion system, cell surface components and regulation system. Phylogenetic analysis revealed genetic relatedness between our isolates and previously sequenced bovine strains from Egypt as well as human strains from other nearby countries in the region., (© 2024. The Author(s).)

Coal, a sedimentary rock harbours a complex microbial community that plays a significant role in its formation and characteristics. However, coal metagenome sequencing and studies were less, limiting our understanding of this complex ecosystem. This study aimed to reconstruct high-quality metagenome-assembled genomes (MAGs) from the coal sample collected in the Neyveli mine to explore the unrevealed diversity of the coal microbiome. Using Illumina sequencing, we obtained high-quality raw reads in FASTQ format. Subsequently, de novo assembly and binning with metaWRAP software facilitated the reconstruction of coal MAGs. Quality assessment using CheckM identified 10 High-Quality MAGs (HQ MAGs), 7 medium-quality MAGs (MQ MAGs), and 6 low-quality MAGs (LQ MAGs). Further analysis using GTDB-Tk revealed four HQ MAGs as known species like Dermacoccus abyssi, Sphingomonas aquatilis, Acinetobacter baumannii, and Burkholderia cenocepacia. The remaining six HQ MAGs were classified as Comamonas, Arthrobacter, Noviherbaspirillum, Acidovorax, Oxalicibacterium, and Bordetella and designated as novel genomes by the validation of digital DNA-DNA hybridization (dDDH). Phylogenetic analysis and further pangenome analysis across the phylogenetic groups revealed a similar pattern with a high proportion of cloud genes. We further analysed the functional potential of these MAGs and closely related genomes using COG. The comparative functional genomics revealed that novel genomes are highly versatile, potentially reflecting adaptations to the coal environment. BlastKOALA was used to conduct a detailed analysis of the metabolic pathways associated with the MAGs. This study highlights the comparative genomic analysis of novel coal genomes with their closely related genomes to understand the evolutionary relationships and functional properties., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Accurate bacterial identification is essential for determining the causative agent of an infection, thus facilitating appropriate treatment and management strategies in both human and animal health contexts. Some species in the Vibrio genus are recognized pathogens, associated with a variety of infections. However, identification of these bacteria is oftentimes controversial. Therefore, we aimed to evaluate different identification approaches in terms of their reliability in distinguishing Vibrio species. To achieve this, we selected a set of 40 Vibrio isolates previously recovered from water and floating plastic samples in a large bay environment and identified them employing MALDI-TOF mass spectrometry, and rrs and pyrH gene sequencing. A subset of isolates was also submitted to whole genome sequencing. Overall, MALDI-TOF was found to be a fast-screening methodology for identification, notably at genus-level. However, for better species discrimination, pyrH gene sequencing stood out as a more reliable tool in contrast to rrs gene sequencing and MALDI-TOF, as corroborated by whole genome sequencing analysis., (© 2024. The Author(s).)

Background: Multi-drug resistant (MDR) Klebsiella pneumoniae is a public health concern due to its presence in Bangladeshi poultry products and its ability to spread resistance genes. This study genetically characterizes a distinct MDR K. pneumoniae isolate from the gut of poultry in Noakhali, Bangladesh, offering insights into its resistance mechanisms and public health impact., Methods: Klebsiella pneumoniae isolates from broiler and layer poultry were identified using biochemical and molecular analyses. Eleven isolates were tested for antibiotic sensitivity and categorized by their Multiple Antibiotic Resistance Index (MARI) profiles. The isolate with the highest MARI was selected for whole-genome sequencing using Illumina technology. The sequencing data were analyzed for genome annotation, pan-genome analysis, genome similarities, sequence type identification, and the identification of genetic determinants of resistance and virulence genes., Result: We identified 10 MARI profiles among 11 K. pneumoniae isolates, with values ranging from 0.64 to 0.94. The highest MARI of 0.94 was found in an isolate from a layer poultry. This isolate's genome, 5401,789 base pairs long with 89.6 % coverage, showed potential inter-species dissemination, as indicated by core genome phylogenetic analysis. It possessed genes conferring resistance to fluoroquinolones, aminoglycosides, β-lactams, folate pathway antagonists, fosfomycin, macrolides, quinolones, rifamycin, tetracyclines, and polymyxins, including colistin., Conclusion: Poultry serve as reservoirs for MDR K. pneumoniae, which can spread to other species and pose significant health risks. Rigorous monitoring of antibiotic use and genetic characterization of MDR bacterial isolates are essential to mitigate this threat., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Bovine Genital Campylobacteriosis (BGC) is caused by Campylobacter fetus subsp. venerealis and is a notifiable disease to the WOAH (World Organisation for Animal Health). For an effective BGC control program, the reliable differentiation of Campylobacter fetus subsp. venerealis (Cfv) from the closely related Campylobacter fetus subsp. fetus (Cff) is required. However, the available molecular C. fetus subspecies identification assays lack sensitivity and specificity to differentiate C. fetus isolates based on their phenotypic or genotypic differences. Furthermore, the current biochemical subspecies identification is not fully congruent with the genomic differentiation of C. fetus strains. In this study, the genome sequences of 41C. fetus strains with well identified subspecies, were analyzed with the large-scale BLAST score ratio (LS-BSR) pipeline to identify Cff and Cfv specific sequences. With this analysis, the asd gene encoding an aspartate-semialdehyde dehydrogenase was identified, which contained a 6-bp Cff-specific sequence, and this 6-bp sequence was absent in the asd gene of Cfv strains. This sequence was used for the development of PCR assays to differentiate Cff and Cfv strains. The C. fetus subspecies identification of the developed asd PCR assays was in full congruence with the genomic classification of strains and are recommended for molecular identification of C. fetus subspecies in BGC control programs. The asd PCR can be assessed on sequenced genomes using a web interface containing the Cfvcatch tool, which includes placement of the tested genome in a phylogenetic tree with reference C. fetus genomes to distinguish the two subspecies and to detect antimicrobial resistance genes., Competing Interests: Declaration of competing interest The author(s) declare that there are no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Objectives: Pseudomonas aeruginosa is the most common pathogen in the bronchiectasis lung, associated with worsened outcomes. P. aeruginosa genomic studies in this context have been limited to single-country, European studies. We aimed to determine strain diversity, adaptation mechanisms, and AMR features to better inform treatment., Methods: P. aeruginosa from 180 bronchiectasis patients in 15 countries, obtained prior to a phase 3, randomised clinical trial (ORBIT-3), were analysed by whole-genome sequencing. Phylogenetic groups and sequence types were determined, and between versus within patient genetic diversity compared using Analysis of Molecular Variance (AMOVA). The frequency of AMR-associated genes and mutations was also determined., Results: A total of 2854 P. aeruginosa isolates were analysed, predominantly belonging to phylogenetic group 1 (83%, n = 2359). Genetic diversity was far greater between than within patients, responsible for >99.9% of total diversity (AMOVA: phylogroup 1: df = 145, P < 0.01). Numerous pathways were under selection, some shared with CF (e.g., motility, iron acquisition), some unique to bronchiectasis (e.g., novel efflux pump PA1874). Multidrug resistance features were also frequent., Conclusions: We present a 10-fold increase in the availability of genomic data for P. aeruginosa in bronchiectasis, highlighting key distinctions with cystic fibrosis and potential targets for future treatments., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Spread of hypervirulent and multi-drug resistant Klebsiella pneumoniae in raw milk is public health concern due to its potential impact on food safety and public health. Therefore, this study investigated antibiotic susceptibility test (AST), antibiotic resistance genes (ARGs), mutations conferring ARGs, virulence factor and plasmid replicons to check prevalence of fosfomycin resistant MDR K. pneumoniae isolated from raw milk samples collected from Saurashtra region of Gujarat, India. K. pneumoniae isolated from raw milk and subjected to disk diffusion assay. From that, MDR along with fosfomycin resistant isolates were analysed for multi locus sequence typing, presence of ARGs, mutations conferring resistance, virulence factors and plasmid replicon types by using its whole genome sequence. Results shows that, among 32 K. pneumoniae, 8 were phenotypically resistant to fosfomycin. As per WGS analysis, 8 MDR isolates were assigned into different sequence types such as ST3321, ST37, ST2715, ST1087, ST3157, ST299 and ST29. Among that, ST37 is well recognized MDR high risk clone reported worldwide and first time reported from raw milk of Saurashtra region of Gujarat, India. ARGs responsible for resistance to fosfomycin (fosA) were found in all 8 isolates. Other ARGs such as blaSHV, kdeA, OqxA, OqxB, dfrA1, sul1, qnrB4, aadA2 and ere(A) were also detected. High diversity of virulence factors was also identified by detection of genes encoding virulence factors related to iron uptake such as entE, fepD, entA, entB, Irp2, fepG, ybtU, ybtP, fepC, ybtA, ybtE, fepB, ybtS, fyuA, ybtQ, ybtT, ybtX, Irp1, adherence such as yagZ/ecpA, yagV/ecpE, yagX/ecpC, yagV/ecpE, ykgK/ecpR and invasion such as fimA, pla, fimC, fimH, fimB, fimE were detected in eight genomes. Mutations in murA, uhpT and glpT conferring a fosfomycin resistance were also present in genomes of 8 K. pneumoniae. IncF was the most common plasmid replicon type detected in all 8 genomes. The study reports high diversity of virulent and multidrug resistant K. pneumoniae in raw milk. Hence, genomic surveillance plans are urgently required for food borne pathogens., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Giant freshwater prawn (Macrobrachium rosenbergii (MR)) is a significant aquafarm species commercially cultured in Taiwan. Intensive farming practices have led to the outbreak of Lactococcus garvieae (LG), which causes Lactococcosis in MR. Recently, LG has re-emerged and the number of mortalities in prawn farms has increased in Taiwan. However, there is no preventative strategy described and a lack of knowledge on virulence factors and pathogenesis from LG in MR. The most virulent strain of L. garvieae from M. rosenbergii was screened in vivo among seven isolates selected for infectivity testing injecting 0.1 mL of 10 8  CFU/mL bacterial concentration. Among the seven isolates screened, L. garvieae 109-6 resulted in 100% mortality within 3 days post-infection. Furthermore, 109-6 L. garvieae LD 50 dosage from in MR was found to be 10 6  CFU/mL. Subsequently, the most virulent strain 109-6 was sequenced using MinIon Nanopore sequencing. Results indicated that the LG genome yielded a protein-coding of 3857 with 59 tRNA and 16 rRNA and no plasmid. Interestingly, the distribution of subsystems in the annotated genome revealed genes related to virulence, defence, and disease among LG 50 genes. Altogether, the virulent strain and its genome data revealed distinctive features of LG, which hinted toward its pathogenicity and could facilitate for better preventive strategies., (© 2024 John Wiley & Sons Ltd.)

Objectives: Integrating pathogen genomic surveillance with bioinformatics can enhance public health responses by identifying risk and guiding interventions. This study focusses on the two predominant Campylobacter species, which are commonly found in the gut of birds and mammals and often infect humans via contaminated food. Rising incidence and antimicrobial resistance (AMR) are a global concern, and there is an urgent need to quantify the main routes to human infection., Methods: During routine US national surveillance (2009-2019), 8856 Campylobacter genomes from human infections and 16,703 from possible sources were sequenced. Using machine learning and probabilistic models, we target genetic variation associated with host adaptation to attribute the source of human infections and estimate the importance of different disease reservoirs., Results: Poultry was identified as the primary source of human infections, responsible for an estimated 68% of cases, followed by cattle (28%), and only a small contribution from wild birds (3%) and pork sources (1%). There was also evidence of an increase in multidrug resistance, particularly among isolates attributed to chickens., Conclusions: National surveillance and source attribution can guide policy, and our study suggests that interventions targeting poultry will yield the greatest reductions in campylobacteriosis and spread of AMR in the US., Data Availability: All sequence reads were uploaded and shared on NCBI's Sequence Read Archive (SRA) associated with BioProjects; PRJNA239251 (CDC / PulseNet surveillance), PRJNA287430 (FSIS surveillance), PRJNA292668 & PRJNA292664 (NARMS) and PRJNA258022 (FDA surveillance). Publicly available genomes, including reference genomes and isolates sampled worldwide from wild birds are associated with BioProject accessions: PRJNA176480, PRJNA177352, PRJNA342755, PRJNA345429, PRJNA312235, PRJNA415188, PRJNA524300, PRJNA528879, PRJNA529798, PRJNA575343, PRJNA524315 and PRJNA689604. Contiguous assemblies of all genome sequences compared are available at Mendeley data (assembled C. coli genomes doi: 10.17632/gxswjvxyh3.1; assembled C. jejuni genomes doi: 10.17632/6ngsz3dtbd.1) and individual project and accession numbers can be found in Supplementary tables S1 and S2, which also includes pubMLST identifiers for assembled genomes. Figshare (10.6084/m9.figshare.20279928). Interactive phylogenies are hosted on microreact separately for C. jejuni (https://microreact.org/project/pascoe-us-cjejuni) and C. coli (https://microreact.org/project/pascoe-us-ccoli)., Competing Interests: Declaration of Competing Interest All authors declare no conflict of interest., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Synechococcus sp. PCC7002 has been considered as a photosynthetic chassis for the conversion of CO 2 into biochemicals through genetic modification. However, conventional genetic manipulation techniques prove inadequate for comprehensive genetic modifications in this strain. Here, we present the development of a genome editing tool tailored for S. PCC7002, leveraging its endogenous type I-D CRISPR-Cas system. Utilizing this novel tool, we successfully deleted the glgA1 gene and iteratively edited the genome to obtain a double mutant of glgA1 and glgA2 genes. Additionally, large DNA fragments encompassing the entire type I-A (∼14 kb) or III-B CRISPR-Cas (∼21 kb) systems were completely knocked-out in S. PCC7002 using our tool. Furthermore, the endogenous pAQ5 plasmid, approximately 38 kb in length, was successfully cured from S. PCC7002. Our work demonstrates the feasibility of harnessing the endogenous CRISPR-Cas system for genome editing in S. PCC7002, thereby enriching the genetic toolkit for this species and providing a foundation for future enhancements in its biosynthetic efficiency., Competing Interests: Declaration of Competing Interest The authors have no conflicts of interest to declare., (Copyright © 2024 Elsevier GmbH. All rights reserved.)

Although diphtheria is a vaccine-preventable disease, numerous cases are still reported around the world, as well as outbreaks in countries, including European ones. Species of the Corynebacterium diphtheriae complex are potentially toxigenic and, therefore, must be considered given the possible consequences, such as the circulation of clones and transmission of antimicrobial resistance and virulence genes. Recently, Corynebacterium rouxii was characterized and included among the valid species of the complex. Therefore, two cases of C. rouxii infection arising from infections in domestic animals are presented here. We provide molecular characterization, phylogenetic analyses, genome sequencing, and CRISPR-Cas analyses to contribute to a better understanding of the molecular bases, pathogenesis, and epidemiological monitoring of this species, which is still little studied. We confirmed its taxonomic position with genome sequencing and in silico analysis and identified the ST-918 for both strains. The clinical isolates were sensitive resistance to benzylpenicillin and rifampin. Antimicrobial resistance genes, including tetB, rpoB2, and rbpA genes, were predicted. The bla and ampC genes were not found. Several virulence factors were also detected, including adhesion, iron uptake systems, gene regulation (dtxR), and post-translational modification (MdbA). Finally, one prophage and the Type I-E CRISPR-Cas system were identified., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

In the prospect of novel potential biocontrol agents, a new strain BDI-IS1 belonging to the recently described Bacillus nakamurai was selected for its strong in vitro antimicrobial activities against a range of bacterial and fungal phytopathogens. Genome mining coupled with metabolomics revealed that BDI-IS1 produces multiple non-ribosomal secondary metabolites including surfactin, iturin A, bacillaene, bacillibactin and bacilysin, together with some some ribosomally-synthesized and post-translationally modified peptides (RiPPs) such as plantazolicin, and potentially amylocyclicin, bacinapeptin and LCI. Reverse genetics further showed the specific involvement of some of these compounds in the antagonistic activity of the strain. Comparative genomics between the five already sequenced B. nakamurai strains showed that non-ribosomal products constitute the core metabolome of the species while RiPPs are more strain-specific. Although the secondary metabolome lacks some key bioactive metabolites found in B. velezensis, greenhouse experiments show that B. nakamurai BDI-IS1 is able to protect tomato and maize plants against early blight and northern leaf blight caused by Alternaria solani and Exserohilum turcicum, respectively, at levels similar to or better than B. velezensis QST713. The reduction of these foliar diseases, following root or leaf application of the bacterial suspension demonstrates that BDI-IS1 can act by direct antibiosis and by inducing plant defence mechanisms. These findings indicate that B. nakamurai BDI-IS1 can be considered as a good candidate for biocontrol of plant diseases prevailing in tropical regions, and encourage further research into its spectrum of activity, its requirements and the conditions needed to ensure its efficacy., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier GmbH.. All rights reserved.)

1-Hexadecene has been detected at a level of mg/L in both influent and effluent of wastewater treatment plants situated in chemical/pharmaceutical industrial parks, which poses a potential threat to the environment. However, few reports are available on aerobic metabolic pathways and microorganisms involved in 1-Hexadecene degradation. In this study, a new strain of 1-Hexadecene-degrading bacteria, Bacillus sp. Hex-HIT36 (HIT36), was isolated from the activated sludge of a wastewater treatment plants located in an industrial park. The physicochemical properties and degradation efficacy of HIT36 were investigated. HIT36 was cultured on a medium containing 1-Hexadecene as a sole carbon source; it was found to remove ∼67% of total organic carbon as confirmed by mass spectrometric analysis of intermediate metabolites. Metabolomic and genomic analysis showed that HIT36 possesses various enzymes, namely, pyruvate dehydrogenase, dihydropolyhydroxyl dehydrogenase, and 2-oxoglutarate-2-oxoiron oxidoreductase (subunit alpha), which assist in the metabolization of readily available carbon source or long chain hydrocarbons present in the growth medium/vicinity. This suggests that HIT36 has efficient long-chain alkane degradation efficacy, and understanding the alkane degradation mechanism of this strain can help in developing technologies for the degradation of long-chain alkanes present in wastewater, thereby assisting in the bioremediation of environment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

Background: Enterobacter species are included among the normal human gut microflora and persist in a diverse range of other environmental niches. They have become important opportunistic nosocomial pathogens known to harbour plasmid-mediated multi-class antimicrobial resistance (AMR) determinants. Global AMR surveillance of Enterobacterales isolates shows the genus is second to Klebsiella in terms of frequency of carbapenem resistance. Enterobacter taxonomy is confusing and standard species identification methods are largely inaccurate or insufficient. There are currently 27 named species and a total of 46 taxa in the genus distinguishable via average nucleotide identity (ANI) calculation between pairs of genomic sequences. Here we describe an Enterobacter strain, ECC3473, isolated from the wastewater of an Australian hospital whose species could not be determined by standard methods nor by ribosomal RNA gene multi-locus typing., Aim: To characterise ECC3473 in terms of phenotypic and genotypic antimicrobial resistance, biochemical characteristics and taxonomy as well as to determine the global distribution of the novel species to which it belongs., Methods: Standard broth dilution and disk diffusion were used to determine phenotypic AMR. The strain's complete genome, including plasmids, was obtained following long- and short read sequencing and a novel long/short read hybrid assembly and polishing, and the genomic basis of AMR was determined. Phylogenomic analysis and quantitative measures of relatedness (ANI, digital DNA-DNA hybridisation, and difference in G+C content) were used to study the taxonomic relationship between ECC3473 and Enterobacter type-strains. NCBI and PubMLST databases and the literature were searched for additional members of the novel species to determine its global distribution., Results: ECC3473 is one of 21 strains isolated globally belonging to a novel Enterobacter species for which the name, Enterobacter adelaidei sp. nov. is proposed. The novel species was found to be resilient in its capacity to persist in contaminated water and adaptable in its ability to accumulate multiple transmissible AMR determinants., Conclusion: E. adelaidei sp. nov. may become increasingly important to the dissemination of AMR., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Background: Bacterial small regulatory RNA (sRNA) plays a crucial role in cell metabolism and could be used as a new potential drug target in the treatment of pathogen-induced disease. However, experimental methods for identifying sRNAs still require a large investment of human and material resources., Methods: In this study, we propose a novel sRNA prediction model called sRNAdeep based on the DistilBERT feature extraction and TextCNN methods. The sRNA and non-sRNA sequences of bacteria were considered as sentences and then fed into a composite model consisting of deep learning models to evaluate classification performance., Results: By filtering sRNAs from BSRD database, we obtained a validation dataset comprised of 2438 positive and 4730 negative samples. The benchmark experiments showed that sRNAdeep displayed better performance in the various indexes compared to previous sRNA prediction tools. By applying our tool to Mycobacterium tuberculosis (MTB) genome, we have identified 21 sRNAs within the intergenic and intron regions. A set of 272 targeted genes regulated by these sRNAs were also captured in MTB. The coding proteins of two genes (lysX and icd1) are implicated in drug response, with significant active sites related to drug resistance mechanisms of MTB., Conclusion: In conclusion, our newly developed sRNAdeep can help researchers identify bacterial sRNAs more precisely and can be freely available from https://github.com/pyajagod/sRNAdeep.git ., (© 2024. The Author(s).)

Although an environmental contaminant, Alcaligenes sp. are now increasingly reported in clinical infections. Here, we present a whole-genome sequence of an extended spectrum beta-lactamase (ESBL) Alcaligenes nematophilus strain EBU-23 encoding beta-lactamase, bla gene, isolated from the stool of a hospitalized infant with acute gastroenteritis in a tertiary hospital in Nigeria. Antibiotics susceptibility test revealed that the isolate was resistant to the β-lactams, cephalosporins, and penicillin, β-lactam combination, and the fluoroquinolones. Genomic analysis exposed the presence of a class A beta-lactam hydrolase gene and multiple multidrug efflux permease which may be responsible for the beta-lactamase and multidrug resistance observed with the isolate. To the best our knowledge, we describe the first whole-genome sequence of a multidrug-resistant A. nematophilus implicated in acute childhood gastroenteritis in Nigeria., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The genus Serratia is a typical red bacterium involved in prodigiosin synthesis. Here, we report the genome sequence of Serratia rubidaea XU1, which was isolated from radiation-contaminated soil in Xinjiang, China. The genome of XU1 is composed of 4,972,898 base pairs with a GC content of 59.25%. The genome sequence contains 4707 genes and encodes 4573 proteins, 79 tRNAs, and 17 rRNAs. The prodigiosin biosynthesis gene cluster was identified and analyzed, showing a sequence similarity of 85.55-96.02% with Serratia rubidaea. After optimizing the biosynthesis process, XU1 was able to achieve a maximum titer of 574 units/cell of prodigiosin at a pH of 7.5 and a temperature of 25 °C for 36 h. Glycerol at 20 g/L and beef extract at 5 g/L were used as the carbon and nitrogen sources, respectively. Prodigiosin extracted from XU1 demonstrated inhibition of Escherichia coli, Staphylococcus aureus, Enterococcus faecalis and Pseudomonas aeruginosa. The availability of the sequenced genome of XU1 will be greatly beneficial and contribute to complementary studies on the biosynthetic mechanisms of prodigiosin., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Brucellosis, a zoonotic disease in Türkiye, which has significant direct and indirect impacts on the healthcare system and livestock. This study, which aimed to investigate the differences among Brucella spp. isolates originating from different regions of Türkiye, for implications for public health and veterinary medicine., Method: Twenty-one isolates from ruminants and two isolates from humans obtained from various regions of Türkiye were utilized in the study. The isolates were identified and biotyped using traditional microbiological procedures, and whole-genome sequencing (WGS) was performed. This was followed by single nucleotide polymorphism (SNP)--based phylogenetic analysis and WGS-based analysis of virulence and resistance genes. Additionally, phenotypic antimicrobial resistance and phage susceptibilities were determined. The obtained data were then compared for concordance, ensuring the validity and reliability of the results., Results: Our study, employing culture methods, polymerase chain reaction (PCR), and WGS analyses, identified 11 Brucella melitensis (bv 3 (n = 9), one each bv 1 and bv 2) and 12 B. abortus (bv 3 (n = 11), bv 9 (n = 1)) isolates All B. abortus isolates were of bovine origin, while the B. melitensis isolates were from sheep (n = 7), goat (n = 1), ram (n = 1), and humans (n = 2). In the whole-genome SNP-based phylogenetic tree, all B. melitensis strains were found to be of the IIb subtype of genotype II associated with the Eastern Mediterranean lineage. Ten different genotypes were identified in the SNP analysis of the isolates, with a maximum SNP difference of 278 and a minimum SNP difference of 4 among these genotypes. According to the WGS-SNP-based phylogenetic tree of B. abortus isolates, they were grouped in clade C1. In the SNP analysis, where ten different genotypes were identified, the SNP difference among these genotypes was a maximum of 316 and a minimum of 6. In the in silico MLST analysis performed with WGS data, B. melitensis isolates were identified as ST8 and ST102 genotypes, while B. abortus isolates were identified as ST2 and ST3 genotypes. The dominant genotypes were ST8 for B. melitensis and ST2 for B. abortus, respectively. Virulence gene analysis conducted based on WGS data of the 23 B. abortus and B. melitensis isolates revealed 43 virulence gene-associated regions in all strains, irrespective of species, host, or isolation year. Although classical resistance-related genes were not detected by WGS-based antimicrobial resistance gene analysis, phenotypic resistance analysis revealed resistance to azithromycin, rifampin, and trimethoprim/sulfamethoxazole in B. abortus and B. melitensis isolates., Conclusion: Both B. melitensis and B. abortus were circulating species in animals and human. The dominant genotypes were ST8 for B. melitensis and ST2 for B. abortus, respectively. All B. melitensis strains were found to be of the IIb subtype of genotype II associated with the Eastern Mediterranean lineage, while B. abortus isolates, they were grouped in clade C1. Further, a comprehensive study with a sufficient number of isolates covering all regions of Türkiye would provide more accurate information about the current epidemiological situation in the country., (© 2024. The Author(s).)

Minimum inhibitory concentration (MIC) denotes the in vitro benchmark indicating the quantity of antibiotic required to inhibit proliferation of specific bacterial strains. Determining MIC values corresponding to the infecting bacterial strain is paramount for tailoring appropriate antibiotic therapy. In the interim between specimen collection and laboratory-derived MIC outcomes, clinicians frequently resort to empirical therapy informed by retrospective analyses. Here introduces two deep learning approaches, a Convolutional Neural Network (CNN)-based model and an Enformer-based model, integrating genomic data of Klebsiella Pneumoniae and molecular structural data of 20 antibiotics to anticipate the MIC value of the bacterium for each antibiotic under consideration. These models demonstrate enhanced raw accuracy over the existing state-of-the-art model, which rely exclusively on genomic data. The CNN-based model achieves a notable 20% increase in raw accuracy while further mirroring the 1-tier accuracy of the state-of-the-art model. Although the Enformer-based model does not quite reach the performance levels of the CNN-based model, it offers an advantage by eliminating the need for arbitrary data processing steps. This streamlining of the data processing pipeline facilitates fast updates and improves the model interpretability. It is expected that these deep learning paradigms can significantly inform and bolster clinician decision-making during the empirical treatment phase., (© 2024. The Author(s).)

The recent emergence of Elizabethkingia anophelis as a human pathogen is a major concern for global public health. This organism has the potential to cause severe infections and has inherent antimicrobial resistance. The potential for widespread outbreaks and rapid global spread highlights the critical importance of understanding the biology and transmission dynamics of this infectious agent. We performed a large-scale analysis of available 540 E. anophelis, including one novel strain isolated from raw milk and sequenced in this study. Pan-genome analysis revealed an open and diverse pan-genome in this species, characterized by the presence of many accessory genes. This suggests that the species has a high level of adaptability and can thrive in a variety of environments. Phylogenetic analysis has also revealed a complex population structure, with limited source-lineage correlation. We identified diverse antimicrobial resistance factors, including core-genome and accessory ones often associated with mobile genetic elements within specific lineages. Mobilome analysis revealed a dynamic landscape primarily composed of genetic islands, integrative and conjugative elements, prophage elements, and small portion of plasmids emphasizing a complex mechanism of horizontal gene transfer. Our study underscores the adaptability of E. anophelis, characterized by a diverse range of antimicrobial resistance genes, putative virulence factors, and genes enhancing fitness. This adaptability is also supported by the organism's ability to acquire genetic material through horizontal gene transfer, primarily facilitated by mobile genetic elements such as integrative and conjugative elements (ICEs). The potential for rapid evolution of this emerging pathogen poses a significant challenge to public health efforts., (© 2024. The Author(s).)

Sequence variation observed in populations of pathogens can be used for important public health and evolutionary genomic analyses, especially outbreak analysis and transmission reconstruction. Identifying this variation is typically achieved by aligning sequence reads to a reference genome, but this approach is susceptible to reference biases and requires careful filtering of called genotypes. There is a need for tools that can process this growing volume of bacterial genome data, providing rapid results, but that remain simple so they can be used without highly trained bioinformaticians, expensive data analysis, and long-term storage and processing of large files. Here we describe split k -mer analysis (SKA2), a method that supports both reference-free and reference-based mapping to quickly and accurately genotype populations of bacteria using sequencing reads or genome assemblies. SKA2 is highly accurate for closely related samples, and in outbreak simulations, we show superior variant recall compared with reference-based methods, with no false positives. SKA2 can also accurately map variants to a reference and be used with recombination detection methods to rapidly reconstruct vertical evolutionary history. SKA2 is many times faster than comparable methods and can be used to add new genomes to an existing call set, allowing sequential use without the need to reanalyze entire collections. With an inherent absence of reference bias, high accuracy, and a robust implementation, SKA2 has the potential to become the tool of choice for genotyping bacteria. SKA2 is implemented in Rust and is freely available as open-source software., (© 2024 Derelle et al.; Published by Cold Spring Harbor Laboratory Press.)

Escherichia coli, a commensal species of the human gut, is an opportunistic pathogen that can reach extra-intestinal compartments, including the bloodstream and the bladder, among others. In non-immunosuppressed patients, purifying or neutral evolution of E. coli populations has been reported in the gut. Conversely, it has been suggested that when migrating to extra-intestinal compartments, E. coli genomes undergo diversifying selection as supported by strong evidence for adaptation. The level of genomic polymorphism and the size of the populations translocating from gut to extra-intestinal compartments is largely unknown. To gain insights into the pathophysiology of these translocations, we investigated the level of polymorphism and the evolutionary forces acting on the genomes of 77 E. coli isolated from various compartments in three immunosuppressed patients. Each patient had a unique strain, which was a mutator in one case. In all instances, we observed that translocation encompasses much of the genomic diversity present in the gut. The same signature of selection, whether purifying or diversifying, and as anticipated, neutral for mutator isolates, was observed in both the gut and bloodstream. Additionally, we found a limited number of non-specific mutations among compartments for non-mutator isolates. In all cases, urine isolates were dominated by neutral selection. These findings indicate that substantial proportions of populations are undergoing translocation and that they present a complex compartment-specific pattern of selection at the patient level.IMPORTANCEIt has been suggested that intra and extra-intestinal compartments differentially constrain the evolution of E. coli strains. Whether host particular conditions, such as immunosuppression, could affect the strain evolutionary trajectories remains understudied. We found that, in immunosuppressed patients, large fractions of E. coli gut populations are translocating with variable modifications of the signature of selection for commensal and pathogenic isolates according to the compartment and/or the patient. Such multiple site sampling should be performed in large cohorts of patients to gain a better understanding of E. coli extra-intestinal diseases., Competing Interests: The authors declare no conflict of interest.

The discovery of severe acute respiratory syndrome-coronavirus-2-like and Middle East respiratory syndrome-coronavirus-like viruses in Malayan pangolins has raised concerns about their potential role in the spread of zoonotic diseases. Herein, we describe the isolation and whole-genome sequencing of potentially zoonotic two bacterial pathogens from diseased Malaysian pangolins ( Manis javanica )- Corynebacterium ulcerans and Erysipelothrix sp. The newly identified species were designated as C. ulcerans P69 and Erysipelothrix sp. P66. C. ulcerans P69 exhibited 99.2% whole-genome nucleotide identity to human bacterial isolate 4940, suggesting that it might have zoonotic potential. Notably, C. ulcerans P69 lacked the diphtheria toxin ( tox ) gene that is widely used in vaccines to protect humans from corynebacterial infection, which suggests that the current vaccine may be of limited efficacy against this pangolin strain. C. ulcerans P69 also contains other known virulence-associated genes such as pld and exhibits resistance to several antibiotics (erythromycin, clindamycin, penicillin G, gentamicin, tetracycline), which may affect its effective control. Erysipelothrix sp. P66 was closely related to Erysipelothrix sp. strain 2-related strains, exhibiting 98.8% whole-genome nucleotide identity. This bacterium is lethal in mice, and two commercial vaccines failed to protect its challenge, such that it could potentially pose a threat to the swine industry. Overall, this study highlights that, in addition to viruses, pangolins harbor bacteria that may pose a potential threat to humans and domestic animals, and which merit attention., Importance: This study firstly reports the presence of two potentially zoonotic bacteria, Corynebacterium ulcerans and Erysipelothrix sp., in diseased Malaysian pangolins collected in 2019. The pangolin C. ulcerans is lethal in mice and resists many antibiotics. It clustered with a lethal human strain but lacked the diphtheria toxin gene. Diphtheria toxin is widely used as a vaccine around the world to protect humans from the infection of corynebacteria. The lack of the tox gene suggests that the current vaccine may be of limited efficacy against this pangolin strain. The pangolin Erysipelothrix sp. is the sister clade of Erysipelothrix rhusiopathiae . It is lethal in mice, and two commercial vaccines failed to protect the mice against challenge with the pangolin Erysipelothrix sp., such that this strain could potentially pose a threat to the swine industry. These findings emphasize the potential threat of pangolin bacteria., Competing Interests: The authors declare no conflict of interest.

Soil microorganisms mediate several biological processes through the secretion of natural products synthesized in specialized metabolic pathways, yet functional characterization in ecological contexts remains challenging. Using culture-independent metagenomic analyses of microbial DNA derived directly from soil samples, we examined the potential of biosynthetic gene clusters (BGCs) from six bacterial communities distributed along an altitudinal gradient of the Andes Mountains in the Atacama Desert. We mined 38 metagenome-assembled genomes (MAGs) and identified 168 BGCs. Results indicated that most predicted BGCs were classified as non-ribosomal-peptides (NRP), post-translational modified peptides (RiPP), and terpenes, which were mainly identified in genomes of species from Acidobacteriota and Proteobacteria phyla. Based on BGC composition according to types of core biosynthetic genes, six clusters of MAGs were observed, three of them with predominance for a single phylum, of which two also showed specificity to a single sampling site. Comparative analyses of accessory genes in BGCs showed associations between membrane transporters and other protein domains involved in specialized metabolism with classes of biosynthetic cores, such as resistance-nodulation-cell division (RND) multidrug efflux pumps with RiPPs and the iron-dependent transporter TonB with terpenes. Our findings increase knowledge regarding the biosynthetic potential of uncultured bacteria inhabiting pristine locations from one of the oldest and driest nonpolar deserts on Earth.IMPORTANCEMuch of what we know about specialized metabolites in the Atacama Desert, including Andean ecosystems, comes from isolated microorganisms intended for drug development and natural product discovery. To complement research on the metabolic potential of microbes in extreme environments, comparative analyses on functional annotations of biosynthetic gene clusters (BGCs) from uncultivated bacterial genomes were carried out. Results indicated that in general, BGCs encode for structurally unique metabolites and that metagenome-assembled genomes did not show an obvious relationship between the composition of their core biosynthetic potential and taxonomy or geographic distribution. Nevertheless, some members of Acidobacteriota showed a phylogenetic relationship with specific metabolic traits and a few members of Proteobacteria and Desulfobacterota exhibited niche adaptations. Our results emphasize that studying specialized metabolism in environmental samples may significantly contribute to the elucidation of structures, activities, and ecological roles of microbial molecules., Competing Interests: The authors declare no conflict of interest.

Background: Hypersaline habitats, as extreme environments, are a great source of well-adapted organisms with unique properties as they have evolved various strategies to cope with these extreme conditions. Bioinformatics and genomic mining may shed light on evolutionary relationships among them. Therefore, the aim of this study was to assess the biodiversity and especially the strategies evolved within the Idiomarina genus, with the primary focus on the taxonomy and genomic adaptations of two novel strains affiliated with Idiomarina genus isolated from unique environment - brines of two Early Miocene salt deposits., Results: Both analyzed species belonged to the Idiomarina loihiensis cluster with similarity levels of 16S rRNA gene sequences as high as 99.5% and showed a significant genome size reduction, known characteristic of Idiomarina genomes, though within the genome of Sol25 strain the lowest extent of the carbohydrate utilization genes reduction was observed t among the Idiomarina species. Moreover, the comparative genome analyses indicated that despite both strains being isolated from geographically and geologically similar environments (brines from at least 12 Ma), the species showed higher relatedness to other Idiomarina species than to each other., Conclusion: The present findings highlighted the importance of genomic data in resolving taxonomic uncertainties and understanding of adaptation strategies of extremophiles. Geographic isolation likely contributed to population divergence of the Idiomarina genus, and the recent study offered insights into biogeographic patterns and allopatric speciation of this bacterial group., (© 2024. The Author(s).)

In response to the growing demand for immune-related products, this study evaluated the safety and immune-modulating potential of three newly discovered Lactiplantibacillus plantarum strains (GKM3, GKK1, and GKD7) through toxicity tests and whole-genome sequencing. Safety evaluations, including the analysis of antimicrobial resistance genes, virulence factors, plasmids, and prophages, classified these strains as safe for human consumption. Acute oral toxicity tests further supported their safety. To evaluate their immune-modulating potential, dendritic cells were exposed to these strains, and the secretion of key cytokines (IFN-β and IL-12) was measured. Among the strains, GKK1 exhibited the highest enhancement of IFN-β and IL-12 production, suggesting its potential as an immune-stimulating probiotic. Bioinformatics analysis revealed potential metabolic pathways and secondary metabolites, including predicted bacteriocins, associated with immune modulation. The presence of a nitrate reductase region in the GKK1 strain indicated its ability to produce nitric oxide, a critical molecule involved in immune regulation and host defense. The presence of glucorhamnan-related gene clusters in GKK1 also suggested immune-enhancing effects. Nitrate reductase expression was confirmed using qPCR, with the highest levels detected in GKK1. Moreover, this study is the first to show an anti-inflammatory effect of plantaricin A, linked to its presence in strain GKM3 and its potential therapeutic applications due to sequence similarity to known anti-inflammatory peptides. Overall, these three L. plantarum strains demonstrated a safe profile and GKK1 showed potential as an immunity-enhancing probiotic. However, additional investigation is required to confirm the involvement of specific metabolic pathways, secondary metabolites, and bacteriocins in immune responses.

The emergence and spread of multidrug-resistance (MDR) pathogenic Escherichia coli due to horizontal gene transfer of antibiotic resistance genes (ARGs) and virulence factors (VFs) is a global health concern, particularly in developing countries. While numerous studies have focused on major sequence types (STs), the implication of minor STs in ARG dissemination and their pathogenicity remains crucial. In this study, two E. coli strains (PEC1011 and PEC1012) were isolated from wild bird feces in Pakistan and identified as ST2178 based on their complete genome sequences. To understand this minor ST, 204 genome assemblies of ST2178 were comparatively analyzed with the isolates' genomes. The phylogenetic analyses revealed five subclades of ST2178. Subclade E strains were predominantly isolated from human specimens, whereas subclades A and B strains including strains PEC1011 and PEC1012, respectively, were frequently isolated from animal. Mobile genetic elements (MGEs) exhibited the positive correlation with ARGs but not with VFs in this ST. Plasmid-borne ARGs exhibited higher correlation with plasmid-borne MGEs, indicating the role of diverse mobile plasmid structures in ARG transmission. Subclade E exhibited diverse plasmid-borne ARG repertoires correlated with MGEs, marking it as a critical surveillance target. In the case of VFs, they exhibited phylogeny-dependent profiles. Strain PEC1012 harbored various plasmid-borne ARGs, which are similar with conserved ARG repertoires in subclade A. The presence of unique ARG insertion in pPEC1012 highlights the importance of subclade A in ARG dissemination. This study comprehensively elucidates the landscape of ST2178, identifying critical phylogenetic subclades and their characteristics in ARG and VF occurrence.

The cell cycle-regulated DNA methyltransferase CcrM is conserved in most Alphaproteobacteria, but its role in bacteria with complex or multicentric genomes remains unexplored. Here, we compare the methylome, the transcriptome and the phenotypes of wild-type and CcrM-depleted Agrobacterium tumefaciens cells with a dicentric chromosome with two essential replication origins. We find that DNA methylation has a pleiotropic impact on motility, biofilm formation and viability. Remarkably, CcrM promotes the expression of the repABCCh2 operon, encoding proteins required for replication initiation/partitioning at ori2, and represses gcrA, encoding a conserved global cell cycle regulator. Imaging ori1 and ori2 in live cells, we show that replication from ori2 is often delayed in cells with a hypo-methylated genome, while ori2 over-initiates in cells with a hyper-methylated genome. Further analyses show that GcrA promotes the expression of the RepCCh2 initiator, most likely through the repression of a RepECh2 anti-sense RNA. Altogether, we propose that replication at ori1 leads to a transient hemi-methylation and activation of the gcrA promoter, allowing repCCh2 activation by GcrA and contributing to initiation at ori2. This study then uncovers a novel and original connection between CcrM-dependent DNA methylation, a conserved epigenetic regulator and genome maintenance in an Alphaproteobacterial pathogen., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

In this study, a novel species within the genus Paracoccus was isolated from the coastal soil of Dokdo (Seodo) Island and investigated. We elucidated the novel species, designated MBLB3053 T , through genomic analysis of novel functional microbial resources. Cells were gram-negative, non-motile, and coccoid, and the colony was light orange in color. Phylogenetic analysis based on the 16S rRNA gene showed that strain MBLB3053 T was related to the genus Paracoccus , with 98.5% similarity to Paracoccus aestuariivivens . Comparative genome analysis also revealed the strain to be a novel species of the genus Paracoccus by average nucleotide identity and in silico DNA-DNA hybridization values. Through secondary metabolite analysis, terpene biosynthetic gene clusters associated with carotenoid biosynthesis were found in strain MBLB3053 T . Using high-performance liquid chromatography, strain MBLB3053 T was confirmed to produce carotenoids, including all- trans -astaxanthin, by comparison to the standard compound. Notably, the isolate was also confirmed to produce carotenoids that other closely related species did not produce. Based on this comprehensive polyphasic taxonomy, strain MBLB3053 T represents a novel species within the genus Paracoccus , for which the name Paracoccus aurantius sp. nov is proposed. The type strain was MBL3053 T (=KCTC 8269 T =JCM 36634 T ). These findings support the research and resource value of this novel species, which was isolated from the Dokdo environmental microbiome.

Whipple disease caused by Tropheryma whipplei a gram-positive bacterium is a systemic disorder that impacts not only the gastrointestinal tract but also the vascular system, joints, central nervous system, and cardiovascular system. Due to the lack of an approved vaccine, this study aimed to utilize immunoinformatic approaches to design multiepitope -based vaccine by utilizing the proteomes of five representative T. whipplei strains. The genomes initially comprised a total of 4,844 proteins ranging from 956 to 1012 proteins per strain. We collected 829 nonredundant lists of core proteins, that were shared among all the strains. Following subtractive proteomics, one extracellular protein, WP&#95;033800108.1, a WhiB family transcriptional regulator, was selected for the chimeric-based multiepitope vaccine. Five immunodominant epitopes were retrieved from the WhiB family transcriptional regulator protein, indicating MHC-I and MHC-II with a global population coverage of 70.61%. The strong binding affinity, high solubility, nontoxicity, nonallergenic properties and high antigenicity scores make the selected epitopes more appropriate. Integration of the epitopes into a chimeric vaccine was carried out by applying appropriate adjuvant molecules and linkers, leading to the vaccine construct having enhanced immunogenicity and successfully eliciting both innate and adaptive immune responses. Moreover, the abilityof the vaccine to bind TLR4, a core innate immune receptor, was confirmed. Molecular dynamics simulations have also revealed the promising potential stability of the designed vaccine at 400 ns. In summary, we have designed a potential vaccine construct that has the ability not only to induce targeted immunogenicity for one strain but also for global T. whipplei strains. This study proposes a potential universal vaccine, reducing Whipple's disease risk and laying the groundwork for future research on multi-strain pathogens., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The emergence of commensalism and mutualism often derives from ancestral parasitism. However, in the case of rhizobium-legume interactions, bacterial strains displaying both pathogenic and nodulation features on a single host have not been described yet. Here, we isolated such a bacterium from Medicago nodules. On the same plant genotypes, the T4 strain can induce ineffective nodules in a highly competitive way and behave as a harsh parasite triggering plant death. The T4 strain presents this dual ability on multiple legume species of the Inverted Repeat-Lacking Clade, the output of the interaction relying on the developmental stage of the plant. Genomic and phenotypic clustering analysis show that T4 belongs to the nonsymbiotic Ensifer adhaerens group and clusters together with T173, another strain harboring this dual ability. In this work, we identify a bacterial clade that includes rhizobial strains displaying both pathogenic and nodulating abilities on a single legume host., (© 2024. The Author(s).)

To address the potential pollution caused by the carcinogen 1, 4-dioxane in aquatic environments, we isolated a highly efficient 1, 4-dioxane-degrading bacterial strain, designated as DXTK-010, from the groundwater contaminated by 1, 4-dioxane. According to the morphological characteristics, the phylogenetic tree established based on the 16S rRNA gene sequence, and the whole genome sequence, we identified DXTK-010 as Aminobacter aminovorans . This strain demonstrated robust degradation capacity within a temperature range of 20 ℃ to 37 ℃ and a pH range of 5.0 to 8.0. Furthermore, single-factor experiments indicated the optimal degradation conditions at 30 ℃ and pH 7.5. Under the optimal conditions, the strain completely degraded 200 mg/L of 1, 4-dioxane within 24 h, achieving a maximum degradation rate of 9.367 mg/(L·h). The Monod equation was adopted to fit the degradation kinetics of 1, 4-dioxane at different initial concentrations, which revealed a maximum specific degradation rate of 0.224 mg 1, 4-dioxane/(mg protein·h), a half-saturation constant ( K s ) of 41.350 mg/L, and a cell yield of 0.130 mg protein/(mg 1, 4-dioxane). Whole genome sequencing revealed a circular chromosome and three plasmids within DXTK-010. Functional gene annotation and analysis underscored the significance of the propane monooxygenase gene cluster and alcohol dehydrogenase gene in facilitating the efficient degradation of 1, 4-dioxane by this strain. DXTK-010 outperformed the existing degraders for 1, 4-dioxane, expanding the strain resources for the bioremediation of 1, 4-dioxane pollution. This study provides a theoretical basis for the practical application of DXTK-010 in the remediation of 1, 4-dioxane pollution.

The accurate reconstruction of genome-scale metabolic models (GEMs) for unculturable species poses challenges due to the incomplete and fragmented genetic information typical of metagenome-assembled genomes (MAGs). While existing tools leverage sequence homology from single genomes, this study introduces pan-Draft, a pan-reactome-based approach exploiting recurrent genetic evidence to determine the solid core structure of species-level GEMs. By comparing MAGs clustered at the species-level, pan-Draft addresses the issues due to the incompleteness and contamination of individual genomes, providing high-quality draft models and an accessory reactions catalog supporting the gapfilling step. This approach will improve our comprehension of metabolic functions of uncultured species., (© 2024. The Author(s).)

Background: Streptococcus pneumoniae is a major cause of mortality globally. The introduction of pneumococcal conjugate vaccines (PCVs) has reduced the incidence of the targeted serotypes significantly, but expansion of non-targeted serotypes, serotype replacement, and incomplete vaccine-targeting contribute to pneumococcal disease in the vaccine era. Here, we characterize the changing population genetic landscape of S. pneumoniae in Norway over a 41-year period (1982-2022)., Methods: Since 2018, all cases of invasive pneumococcal disease have undergone whole-genome sequencing (WGS) at the Norwegian Institute of Public Health. In order to characterize the changing population over time, historical isolates were re-cultured and sequenced, resulting in a historical WGS dataset. Isolates were assigned to global pneumococcal sequence clusters (GPSCs) using PathogenWatch and assigned to serotypes using in silico (SeroBA) and in vitro methods (Quellung reaction). Temporal phylogenetic analyses were performed on GPSCs of particular interest., Results: The availability of WGS data allowed us to study capsular variation at the level of individual lineages. We detect highly divergent fates for different GPSCs following the introduction of PCVs. For two out of eight major GPSCs, we identified multiple instances of serotype switching from vaccine types to non-vaccine types. Dating analyses suggest that most instances of serotype switching predated the introduction of PCVs, but expansion occurred after their introduction. Furthermore, selection for penicillin non-susceptibility was not a driving force for the changing serotype distribution within the GPSCs over time., Conclusions: PCVs have been major shapers of the Norwegian disease-causing pneumococcal population, both at the level of serotype distributions and the underlying lineage dynamics. Overall, the introduction of PCVs has reduced the incidence of invasive disease. However, some GPSCs initially dominated by vaccine types escaped the effect of vaccination through expansion of non-vaccine serotypes. Close monitoring of circulating lineages and serotypes will be key for ensuring optimal vaccination coverage going forward., (© 2024. The Author(s).)

DNA transfer is ubiquitous in the human gut microbiota, especially among species of the order Bacteroidales. In silico analyses have revealed hundreds of mobile genetic elements shared between these species, yet little is known about the phenotypes they encode, their effects on fitness, or pleiotropic consequences for the recipient's genome. In this work, we show that acquisition of a ubiquitous integrative conjugative element (ICE) encoding a type VI secretion system (T6SS) shuts down the native T6SS of Bacteroides fragilis . Despite inactivating this T6SS, ICE acquisition increases the fitness of the B. fragilis transconjugant over its progenitor by arming it with the new T6SS. DNA transfer causes the strain to change allegiances so that it no longer targets ecosystem members with the same element yet is armed for communal defense.

Salmonella enterica, a prominent foodborne pathogen, contributes significantly to global foodborne illnesses annually. This species exhibits significant genetic diversity, potentially impacting its infectivity, disease severity, and antimicrobial resistance. Whole genome sequencing (WGS) offers comprehensive genetic insights that can be utilized for virulence assessment. However, existing bioinformatic tools for studying Salmonella virulence have notable limitations. To address this gap, a Salmonella Virulence Database with a non-redundant, comprehensive list of putative virulence factors was constructed. Two bioinformatic analysis tools, Virulence Factor Profile Assessment and Virulence Factor Profile Comparison tools, were developed. The former provides data on similarity to the reference genes, e-value, and bite score, while the latter assesses the presence/absence of virulence genes in Salmonella isolates and facilitates comparison of virulence profiles across multiple sequences. To validate the database and associated bioinformatic tools, WGS data from 43,853 Salmonella isolates spanning 14 serovars was extracted from GenBank, and WGS data previously generated in our lab was used. Overall, the Salmonella Virulence database and our bioinformatic tools effectively facilitated virulence assessment, enhancing our understanding of virulence profiles among Salmonella isolates and serovars. The public availability of these resources will empower researchers to assess Salmonella virulence comprehensively, which could inform strategies for pathogen control and risk evaluations associated with human illnesses., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Escherichia coli exhibit extensive genetic diversity at the genome level, particularly within their accessory genome. The tRNA integrated genomic islands (GIs), a part of the E. coli accessory genome, play an important role in pathogenicity. However, studies examining the evolution of GIs have been challenging due to their large size, considerable gene content variation and fragmented assembly in draft genomes. Here we examined the evolution of the GI integrated at pheV-tRNA (GI-pheV), with a primary focus on uropathogenic E. coli (UPEC) and the globally disseminated multidrug resistant ST131 clone. We show the gene content of GI-pheV is highly diverse and arranged in a modular configuration, with the P4 integrase encoding gene intP4 the only conserved gene. Despite this diversity, the GI-pheV gene content displayed conserved features among strains from the same pathotype. In ST131, GI-pheV corresponding to the reference strain EC958 (EC958&#95;GI-pheV) was found in ~90% of strains. Phylogenetic analyses suggested that GI-pheV in ST131 has evolved together with the core genome, with the loss/gain of specific modules (or the entire GI) linked to strain specific events. Overall, we show GI-pheV exhibits a dynamic evolutionary pathway, in which modules and genes have evolved through multiple events including insertions, deletions and recombination., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Nhu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)