- Volume 11, Issue 4, 2025

Microbial biostimulants (MBs) offer a sustainable approach to agriculture by helping to reduce reliance on synthetic fertilizers. However, as MBs are intentionally released into the environment, their safety should be rigorously assessed. While taxa with qualified presumption of safety (QPS) benefit from established safety indications, non-QPS taxa lack such guidance. To address this gap, we propose a pipeline combining whole genome sequencing (WGS) and extensive literature search (ELS) data to evaluate microbial safety. We analysed public genomes of three QPS species (Rhodopseudomonas palustris, Bacillus velezensis, Priestia megaterium) and four non-QPS genera (Arthrobacter, Azotobacter, Azospirillum, Herbaspirillum), screening them for virulence factors (VFs), antimicrobial resistance (AMR) genes and mobile genetic elements (MGEs). Results confirmed the safety of QPS taxa, revealing no VFs and only a few intrinsic and non-clinically relevant AMRs. Among non-QPS taxa, VF hits were more prevalent in Azotobacter and Azospirillum spp., though they were mostly related to beneficial plant interactions rather than pathogenicity. AMR genes in non-QPS taxa were primarily associated with efflux pumps or were sporadically distributed. Notably, the only genus-wide pattern observed was that most Azospirillum and Herbaspirillum genomes harboured chromosomally encoded β-lactamases sharing similar genetic structures; however, the detected β-lactamase (bla) genes were distantly related to clinically relevant bla variants, and the absence of MGEs suggests a low risk of horizontal gene transfer, indicating the overall safety of these genera. In general, this WGS–ELS framework provides a robust tool for assessing the safety of non-QPS MBs, supporting regulatory decision-making and ensuring their safe use in sustainable agriculture while safeguarding public health.

Vibrio are widely distributed in aquatic environments and are major pathogens commonly found in aquaculture environments, playing a significant role in human production activities and maintaining ecological stability. Here, a novel phage, vB_VneS_J26, which infects Vibrio neocaledonicus, was isolated from coastal seawater in Qingdao, China. Transmission electron microscopy revealed that vB_VneS_J26 exhibits siphovirus morphotype, with a linear double-stranded DNA genome of 82,477 bp in length and G+C content of 45.11 mol%, encoding 122 putative ORFs. Three auxiliary metabolic genes related to carbon metabolism and host cell redox processes were identified, including a pyruvate phosphate dikinase, which catalyses the reversible conversion between phosphoenolpyruvate and pyruvate and is rarely detected in viruses. Whole-genome phylogenetic and comparative genomic analyses suggested that vB_VneS_J26 represents a potential novel viral family, comprising six isolated vibriophages, proposed as Modirecodeviridae. Phylogeographic analysis indicated that Modirecodeviridae is primarily distributed in epipelagic and mesopelagic zones of the Arctic and temperate tropical oceans.

Streptococcus pneumoniae is a significant cause of bacterial infections, including pneumonia, meningitis and septicemia, primarily affecting children, the elderly and immunocompromised individuals. This study aimed to elucidate the serotype and lineage distribution and molecular mechanisms underlying pneumococcal invasiveness through a comprehensive pangenomic analysis of 1416 isolates from Malawi. Our analysis comprised 810 isolates from asymptomatic carriers and 606 isolates from patients with bacteraemia or meningitis. We identified 58 serotypes, with serotypes 1, 5 and 12F exhibiting significantly higher prevalence among patients. These serotypes likely exhibit reduced nasopharyngeal colonization and demonstrate rapid dissemination to sterile sites. Notably, these serotypes form a distinct lineage with distinct genomic characteristics, including the absence of V-type ATP synthase. The pangenome analysis revealed two highly conserved surface protein complexes, F-type ATP synthase and SecA1-SecY, which deserve further investigation as potential targets for novel therapeutic interventions.

Antimicrobial peptides (AMPs) are often produced by eukaryotes to control bacterial populations in both pathogenic and mutualistic symbioses. Several pathogens and nitrogen-fixing legume symbionts depend on transporters called SbmA (or BacA) or BclA (BacA-like) to survive exposure to AMPs. However, how broadly these transporters are distributed amongst bacteria, and their evolutionary history, is poorly understood. We used computational approaches, including phylogenetic and sequence similarity analyses, to examine the distribution of SbmA/BacA and BclA proteins across 1,255 species spanning the domain Bacteria, leading to the identification of 71 and 177 SbmA/BacA and BclA proteins, respectively. In vitro sensitivity assays using legume AMPs and several BclA proteins confirmed that AMP transport is a common feature of BclA homologues. Our analyses indicated that SbmA/BacA homologues are encoded only by species in the phylum Pseudomonadota and are primarily found in just two orders: Hyphomicrobiales and Enterobacterales. BclA homologues are somewhat more broadly distributed and were found in clusters across four phyla. These included several orders of the phyla Pseudomonadota and Cyanobacteriota, the order Mycobacteriales (phylum Actinomycetota) and the class Negativicutes (phylum Bacillota). Many of the clades enriched for species encoding SbmA/BacA or BclA homologues are rich in species that interact with eukaryotic hosts in mutualistic or pathogenic interactions. These observations suggest that SbmA/BacA and BclA proteins have been repeatedly co-opted to facilitate associations with eukaryotic hosts by allowing bacteria to cope with host-encoded AMPs.

The congruence between host and symbiont phylogenies reflects the evolutionary links among ecologically important interactions. As potential key symbionts, the members affiliated to the family Endozoicomonadaceae have previously been investigated for the cophylogenetic relationship with their hosts using their 16S rRNA gene sequences. However, this approach neglects the genomic features of symbionts that may influence the long-term associations between Endozoicomonadaceae members and their hosts. Here, we collected available high-quality genomes of Endozoicomonadaceae from diverse hosts and investigated their genomic features, including genome size, phages, insertion elements and the composition of functional genes. We also tested the host–Endozoicomonadaceae cophylogeny and examined the correlation between the cophylogenetic squared residuals and the genomic features of Endozoicomonadaceae members. Our results revealed a cophylogenetic pattern between members of the Endozoicomonadaceae family and their various hosts. Moreover, we found that the investigated genomes of Endozoicomonadaceae members were differentially eroded by phages and insertion elements. Additionally, Endozoicomonadaceae members with smaller, more eroded genomes tended to exhibit lower cophylogenetic residuals with their hosts. Gene function analysis further revealed that Endozoicomonadaceae members with closer associations with their hosts carried specific genes related to infection processes and host–symbiont interactions. This study suggests that the genomic features of Endozoicomonadaceae members may influence long-term host–Endozoicomonadaceae intimate associations.

Bacterial secondary metabolites are crucial bioactive compounds with significant therapeutic potential, playing key roles in ecological processes and the discovery of novel antimicrobial agents and natural products. Cenotes, as extreme environments, harbour untapped microbial diversity and hold an interesting potential as sources of novel secondary metabolites. While research has focused on the fauna and flora of cenotes, the study of their microbial communities and their biosynthetic capabilities remains limited. Advances in metagenomics and genome sequencing have greatly improved the capacity to explore these communities and their metabolites. In this study, we analysed the microbial diversity and biotechnological potential of micro-organisms inhabiting sediments from a coastal cenote. Metagenomic analyses revealed a rich diversity of bacterial and archaeal communities, containing several novel biosynthetic gene clusters (BGCs) linked to secondary metabolite production. Notably, polyketide synthase BGCs, including those encoding ladderanes and aryl-polyenes, were identified. Bioinformatics analyses of these pathways suggest the presence of compounds with potential industrial and pharmaceutical applications. These findings highlight the biotechnological value of cenotes as reservoirs of secondary metabolites. The study and conservation of these ecosystems are essential to facilitate the discovery of new bioactive compounds that could benefit various industries.

Oral microbiome dysbiosis has been proposed as a potential contributing factor to rising rates of diabetes in pregnancy, with oral health previously associated with an increased risk of numerous chronic diseases and complications in pregnancy, including gestational diabetes mellitus (GDM). However, whilst most studies examining the relationship between GDM and the oral microbiome identify significant differences, these differences are highly variable between studies. Additionally, no previous research has examined the oral microbiome of women with pre-existing type 2 diabetes mellitus (T2DM), which has greater risks of complications to both mother and baby. In this study, we compared the oral microbiome of 11 pregnant women with pre-existing T2DM with 28 pregnant normoglycaemic controls. We used shotgun metagenomic sequencing to examine buccal swab and saliva rinse samples at two time points between 26 and 38 weeks of gestation. To reduce variation caused by the choice of differential abundance analysis tool, we employed a consensus approach to identify differential taxa and pathways due to diabetes status. Differences were identified at the late time point only. In swab samples, there was increased Flavobacteriaceae, Capnocytophaga, Capnocytophaga gingivalis SGB2479, Capnocytophaga leadbetteri SGB2492 and Neisseria elongata SGB9447 abundance in T2DM as well as increased Shannon diversity and richness. In rinse samples, there was an increased abundance of Haemophilus, Pasteurellaceae, Pasteurellales and Proteobacteria. In contrast to studies of the oral microbiome in T2DM or GDM that use a single differential abundance analysis tool, our consensus approach identified few differences between pregnant women with and without T2DM.

Dairy fermentations using mesophilic starter cultures rely on the activity of specific lactic acid bacteria (LAB) such as Lactococcus lactis and Lactococcus cremoris for the acidification of milk. This biotechnological process can be affected by bacteriophage infection of LAB starter strains, which may result in delayed or even failed fermentations. Most studied lactococcal phages commence infection with the binding of a tail-associated receptor-binding protein (RBP) to a host cell surface-exposed cell wall polysaccharide (CWPS). In the present study, phage prevalence and diversity in whey samples originating from fermentations performed in various European countries employing undefined mesophilic starter cultures were investigated using phageome analysis. The range of Skunavirus RBP genotypes present in the phageomes and associated RBP-CWPS binding abilities were evaluated, resulting in the refinement and expansion of the Skunavirus RBP grouping system and the identification of several heretofore unknown Skunavirus RBP (sub)groups. These findings substantially expand our knowledge on lactococcal Skunavirus RBP diversity and their binding specificity towards CWPS receptor structures, thereby improving the predictability of fermentation outcomes and robustness of starter culture rotations and blends.

Escherichia albertii is a Gram-negative facultative anaerobic bacterium that causes diarrhoea in humans. This study shows the isolation of E. albertii from hospitalized paediatric diarrhoeal cases and genome-based characteristics with putative virulence factors and antimicrobial resistance. E. albertii isolates were identified by species-specific PCR, targeting the gene encoding cytolethal distending toxin (Ea-cdt). The genome of E. albertii was sequenced to identify (i) genes encoding virulence factors (ii) antibiotic resistance-encoding genes, including the mobile genetic elements and (iii) core gene-based phylogenetic relationships and pan-genome features. A total of 10 (1.2%) E. albertii isolates were isolated from 854 faecal samples, of which 6 (60%) were found as the sole pathogen and the remaining 4 (40%) were identified along with other pathogens, such as enteroaggregative Escherichia coli, rotavirus and adenovirus. Patients from whom E. albertii was isolated presented cholera-like diarrhoea, i.e. with watery stool (60%) with moderate dehydration (100%), fever (20%) and abdominal pain (20%). The antimicrobial susceptibility testing of E. albertii showed that most of the isolates were susceptible or reduced susceptible to most of the antibiotics except resistance to erythromycin (80%), tetracycline (50%), nalidixic acid (40%), ampicillin (40%), doxycycline (30%) and ceftriaxone (20%). In the whole-genome sequence, E. albertii isolates revealed several virulence-encoding genes, namely the intimin (eae, E. coli attaching and effacing), the cytolethal distending toxin type II subunit A (cdt-IIA), adhesion (paa, porcine attaching- and effacing-associated), non-LEE (locus of enterocyte effacement) encoded effector A (nleA) and antimicrobial resistance genes (ARGs) conferring resistance to tetracycline (tetA, tetR), sulphonamides (sul2), fluoroquinolones (qnrS) and beta-lactamases (bla CTX-M, blaTEM). The SNP-based phylogenetic analysis of 647 whole genomes of E. albertii isolates from the National Center for Biotechnology Information databases did not reveal any comparable clustering pattern based on the biological source and place of isolation. The genome of some of the E. albertii was closely related to those of the isolates from China and the United Kingdom. The PFGE patterns revealed that most of the E. albertii isolates were distinct clones. This study reports on the extensive genome analysis of diarrhoea-associated E. albertii harbouring multiple virulence and ARGs.

Klebsiella pneumoniae (Kp), a ubiquitous pathogen found in diverse ecological niches, poses a threat to human and animal health. Hypervirulent Kp (hvKp) is concerning for its acquisition of virulence and antimicrobial resistance genes through plasmids. This study investigates hvKp as a cause of septicaemia in piglets in the Netherlands and examines the role of plasmids in virulence and host association. We collected 41 Kp isolates cultured from necropsies submitted from 15 different farms (2013–2020) and sequenced them using long-read sequencing. We identified sequence type (ST) 25 as the dominant Kp (67%, 10/15 farms) associated with septicaemia in pigs in the Netherlands. ST25 isolates displayed a hypervirulent profile, including the K2 hyper-capsule type and carried an iuc3 virulence plasmid. Further analysis revealed two ST25 clonal groups: CG25 and CG3804, a novel porcine clone. Multidrug resistance was identified in CG25 isolates from five pig farms. There was one colistin-resistant isolate carrying mcr-1 on a plasmid. Comparative genomic analysis was performed by including a large dataset of related publicly available Kp genomes from ST25 humans (n=230) and pigs (n=12) of all STs for phylogenetic and plasmid analysis. Pangenomic analysis revealed significantly higher iuc3 prevalence in global CG25 pig isolates (98%, 40/41) compared to humans (10%, 24/234) correlating with their enhanced virulence (scores 3–4 vs 0–1). The study highlights ST25 hvKp causing septicaemia in piglets in the Netherlands for the first time. Aerobactin lineage iuc3 on a plasmid is associated with infections in pigs and is responsible for an increased virulence score.

Staphylococci are the most common cause of orthopaedic device-related infections (ODRIs), with Staphylococcus aureus responsible for a third or more of cases. This prospective clinical and laboratory study investigated the association of genomic and phenotypic variation with treatment outcomes in ODRI isolates. Eighty-six invasive S. aureus isolates were collected from patients with ODRI, and clinical outcome was assessed after a follow-up examination of 24 months. Each patient was then considered to have been ‘cured’ or ‘not cured’ based on predefined clinical criteria. Whole-genome sequencing and molecular characterization identified isolates belonging to globally circulating community- and hospital-acquired lineages. Most isolates were phenotypically susceptible to methicillin and lacked the staphylococcal cassette chromosome mec cassette [methicillin-susceptible S. aureus (MSSA); 94%] but contained several virulence genes, including toxins and biofilm genes. Whilst recognizing the role of the host immune response, we identified genetic variance, which could be associated with the infection severity or clinical outcome. Whilst this and several other studies reinforce the role antibiotic resistance [e.g. methicillin-resistant S. aureus (MRSA) infection] has on treatment failure, it is important not to overlook MSSA that can cause equally destructive infections and lead to poor patient outcomes.

Salmonella enterica serotype Enteritidis (SE) is a common foodborne pathogen that can cause human salmonellosis. Identifying closely related cases is essential to control the pathogen through, e.g. outbreak investigation, but it is often challenging due to the low genetic diversity of SE, particularly with traditional typing methods. This study aimed to investigate the population structure of SE genomes collected during routine surveillance in the Netherlands using whole-genome sequencing (WGS), their clustering, temporal distribution and the association between epidemiological and phenotypic antimicrobial resistance (AMR) factors and the persistence of SE clusters. We also investigated the distribution of genotypic AMR markers among these isolates. The study collection comprised 1,669 unique SE isolates from human infections collected from Dutch surveillance between 2019 and 2023, and their relatedness was derived using core-genome multi-locus sequence typing and Hamming distances. Based on the results, the 216 clusters comprised 1,085 sequences, in addition to 584 sequences depicted as singletons. These clusters predominantly fell within three major lineages, of which two were the previously described Global and Atlantic lineages. Of these clusters, approximately a third persisted for more than 1 year during the 5-year study period. However, no statistically significant associations were found between epidemiological factors, such as age, gender and travel history, or phenotypic AMR and the persistence of SE clusters. The most common AMR genetic markers observed were related to antimicrobial classes of (fluor)quinolones, β-lactamases and aminoglycosides. This study provides a better understanding of the genomic epidemiology of SE in the Netherlands based on WGS. Further analysis that includes samples from the food-chain supply, along with higher resolution methods during a post-Coronavirus Disease of 2019 (COVID-19) period, may provide more insights into the possible causes of the persistence of SE clusters.

Group B Streptococcus (GBS) is a commensal bacterium that can cause severe infections in infants and adults with comorbidities. Resistance and reduced susceptibility to antibiotics are continually on the rise, and vaccines remain in development. Prophages have been reported to contribute to GBS evolution and pathogenicity. However, no studies are available to date on prophage contribution to the epidemiology of GBS isolates from humans in South America. In the context of an Argentinian multicentric study, we had previously phenotypically characterized 365 human GBS isolates from invasive disease, urinary infections and maternal colonization. These isolates had been whole-genome sequenced, and their prophage presence was bioinformatically determined. In this study, we genomically characterized the isolates and analysed the prophage content in the context of the epidemiological data. The phylogenetic analysis of the 365 genomes with 103 GBS from public databases revealed that Argentinian GBS were related to isolates from around the world. The most prevalent lineages, independent of the isolated source, were CC23/Ia and CC12/Ib. Genes encoding virulence factors involved in immune response evasion, tissue damage and adherence to host tissues, and invasion were found in all of the genomes in accordance with previously described lineage distribution. According to the prevalent capsular types and the distribution of specific virulence factors in Argentinian GBS, over 95% coverage would be expected from the vaccines currently under development. Antibiotic resistance determinants (ARDs) to at least one antibiotic class were found in 90% of the genomes, including novel mutations in pbp2x, while more than 15% carried ARDs to three or more classes. GBS collected from urinary infections carried a significantly higher proportion of ARDs to multiple antibiotic classes than the rest of the isolates. A total of 454 prophages were found among the 468 genomes analysed, which were classified into 23 prophage types. Prophage presence exhibited variations based on GBS clonal complex and capsular type. A possible association between an increased GBS pathogenicity and the carriage of prophages with integrase type GBSInt8 and/or the presence of genes that encode the Phox Homology domain has been observed. The highest prevalence of prophages per genome was found in lineages CC17/III and CC19/III, while the lowest amount was observed in CC12/Ib. Overall, the highest density of prophages, virulence factors and ARDs determinants was found in CC19 isolates, mostly of capsular type III, independent of the isolates’ source. This is the first analysis of the human-associated GBS population in South America based on whole-genome sequencing data, which will make a significant contribution to future studies on the global GBS population structure.

Talaromyces marneffei is a dimorphic fungus that transitions from a filamentous form at 25 °C to a pathogenic yeast form at 37 °C, demonstrating pathogenicity mostly in immunocompromised individuals, such as those with human immunodeficiency virus/AIDS. Though it is one of the most severe infectious fungi in Southeast Asia, the lack of comprehensive genomic analysis has hindered advancement in strain differentiation, diagnosis and treatment. In this study, we assembled a high-quality genome of T. marneffei ATCC 18224, resulting in a 28.9 Mb genome distributed across 11 contigs, using third-generation Oxford Nanopore Technologies sequencing reads. Notably, we identified a strain-specific 740-kb segmental duplication in strain ATCC 18224, potentially mediated by inserting a Ty1/Copia long terminal repeat (LTR) retrotransposon. This segmental duplication includes various functional genes, with 75 differentially expressed during its dimorphic transition. Comparative genomic analysis revealed large-scale rearrangements in strains PM1 and 11CN-20-091, which were inconsistent with the phylogenomic trees of six T. marneffei strains and required further investigation. Additionally, we observed substantial genetic structural variations in LTR retrotransposons, particularly within the Ty1/Copia family, including two significant recent expansions in strain ATCC 18224. In summary, the identification and characterization of these extensive genomic structural variations in T. marneffei contribute to a deep understanding of its genetic diversity and will facilitate improvements in genotyping, classification and genomic surveillance.

Phytoplasmas (genus ‘Candidatus Phytoplasma’) encompass a group of uncultivated bacteria affecting numerous plant species and causing significant damage in agriculture worldwide. They have a dual parasitic cycle, including colonization of both plant phloem and insect cells. Their genomes are small, diverse, repetitive, prone to rearrangements and harbour transposon-like elements known as potential mobile units (PMUs). In the Euro-Mediterranean region, ‘Ca. P. solani’ is an important species due to its broad range of plant hosts and insect vectors. To provide insights into the genomic diversity of this species, particularly the repertoire of putative effectors and PMUs, this study conducted genome sequencing and analyses of two ‘Ca. P. solani’ strains originating from different plants and transmitted by different insects. Based on de novo assembly, we obtained 19 contigs totalling 656 141 bp for strain STOL and 28 contigs totalling 707 036 bp for strain ST19. The prevalence of repetitive sequences and PMUs contributed to the fragmentation of these draft assemblies. The annotation identified 28 and 26 genes that encode putative secreted proteins in these two strains, respectively, including several homologues of previously characterized phytoplasma effectors. Our comparative analyses further identified species- and strain-specific genes. Frequently, genes that encode putative secreted proteins and effectors were found within PMU-like regions in both genomes. Moreover, strain STOL showed characteristics of a more reduced genome, having fewer PMU-like repetitive elements and genome rearrangements, while strain ST19 exhibited a higher level of sequence divergence in its PMU genes. The high levels of genomic diversity among ‘Ca. P. solani’ strains suggested rapid evolution of this species, which may contribute to its wide host range and adaptability potential. This study provides novel data on the diversification of ‘Ca. P. solani’ genomes. These results provide a foundation for future functional studies of putative effectors and their interactions with host targets, which could facilitate deciphering the pathogenicity strategies of this successful and versatile pathogen.

Listeria monocytogenes is a bacterial pathogen found in an increasing number of food categories, potentially reflecting an expanding niche and food safety risk profile. In the UK, Listeria monocytogenes sequence type (ST) 121 is more frequently isolated from foods and food environments than from cases of clinical listeriosis, consistent with a relatively low pathogenicity. In this study, we determined the evolution associated with the environmental persistence of a Listeria monocytogenes clone by investigating clone-specific genome features in the context of the ST121 population structure from international sources. To enable unambiguous comparative genomic analysis of ST121 strains, we constructed 16 new high-quality genome assemblies from Listeria monocytogenes isolated from foods, food environments and human clinical sources in the UK from 1987 to 2019. Our dataset was supplemented with additional UK and international genomes from databases held by the Institut Pasteur and the UK Health Security Agency. Time-scaled phylogenetic reconstruction revealed that clade-specific microevolution correlated with key characteristics that may confer adaptations important for success in the environmental niche. For example, a prophage designated LP-13-6 unique to a clade is associated with multi-year persistence in a food production setting. This prophage, observed in a strain that persisted for over a decade, may encode mechanisms facilitating environmental persistence, including the exclusion of other bacteriophages. Pangenome analysis provided insights into other candidate genetic elements associated with persistence and biocide tolerance. The comparative genomic dataset compiled in this study includes an international collection of 482 genome sequences that serve as a valuable resource for future studies to explore conserved genes, regulatory regions, mutations and variations associated with particular traits, such as environmental persistence, pathogenicity or biocide tolerance.