Aug 2026· One Health Advances· Vol 4· 0 citations· 40 references
TL;DR
Together, these findings reveal a previously underappreciated class- and host-dependent organization of the streptococcal mobilome and its AMR cargo.
Abstract
Mobile genetic elements (MGEs) drive genome plasticity, horizontal gene transfer, and antimicrobial resistance (AMR) dissemination in Streptococcus, yet genus-wide comparisons across major MGE classes remain limited. Here, we analyzed 1961 complete chromosomes from 60 Streptococcus species together with 225 plasmids, and compared integrative and conjugative elements (ICEs), integrative and mobilizable elements (IMEs), prophages, and plasmids with respect to host distribution, boundary-supported integration-site preference, mobility-associated modules, representative backbones, and AMR cargo. We identified 1172 ICEs, 2362 IMEs, 3397 prophages, and 225 plasmids, and found that the streptococcal mobilome was strongly partitioned by host lineage, with ICEs and IMEs enriched in Streptococcus dysgalactiae, prophages in Streptococcus pyogenes, and plasmids in Streptococcus suis. Integrated MGEs also displayed class-specific hotspot hierarchies after boundary inspection: ICEs were highly concentrated at rplL, with secondary hotspots at rlmD and rpmH; IMEs were dominated by tRNA-associated sites together with rpsI and rpmG, whereas prophages occupied a broader hotspot spectrum centered on tRNA, rpmE, rpsD, hlpA, and mutL. Mobility analyses further distinguished the classes, showing that ICEs retained a narrow repertoire of conjugative backbones dominated by typeFATA, IMEs displayed the broadest relaxase diversity, and plasmids were mainly non-mobile or mobilizable. Representative family analyses resolved recurrent backbone types within each class, whereas AMR cargo was concentrated in ICEs and plasmids and was further stratified by host species. Together, these findings reveal a previously underappreciated class- and host-dependent organization of the streptococcal mobilome and its AMR cargo.
Streptococcus suis is a major porcine pathogen and a zoonotic agent that causes meningitis and septicemia in humans. Streptococcus parasuis, a recently recognized close relative, remains poorly characterized with regard to its clinical significance and genomic features. In this study, we generated a single-contig closed genome assembly with genome-wide DNA methylation profiles for S. parasuis strain A1, isolated from a diseased pig in Xinjiang, China, and complemented in silico genomic predictions with isolate-level experimental validation of antimicrobial resistance (AMR) genotypes, virulence genotypes, and phenotypic susceptibility for this reference strain. Using this high-quality genome as a reference anchor, we performed comparative genomic analyses across 195 streptococcal genomes, comprising 15 S. parasuis and 180 S. suis strains, to distinguish genome-level co-occurrence of resistance and virulence determinants from their physical colocalization on the same mobile genetic element (MGE).Species boundaries remained clearly delineated at the genomic level, with a median interspecies average nucleotide identity (ANI) of approximately 86.0%, compared with intraspecies ANI medians of 97.5% for S. parasuis and 96.2% for S. suis. Pangenome analysis identified 12,693 gene clusters, of which 1086 were core clusters, and functional annotation revealed significant differences in accessory gene repertoires between the two species. Within this stable genomic framework, S. parasuis genomes carried a higher AMR gene burden; strain A1 harbored 10 AMR genes, multiple virulence-associated genes, three genomic islands, and eight prophage regions. For strain A1, PCR validation confirmed six AMR genes and six virulence genes, and disk diffusion testing demonstrated a multidrug-resistant phenotype consistent with the genotypic profile.Among 235 predicted mobile elements, 19 harbored AMR genes and seven carried Virulence Factor Database (VFDB) homologs, but none carried both categories simultaneously. This finding reflects a lack of detectable same-MGE colocalization under the applied annotation and assembly framework; it should not be interpreted as evidence of biological physical decoupling. Under a random-placement model, the expected number of co-carrying regions was only 0.57, and the probability of observing zero co-carrying regions was P = 0.55. This negative result should be interpreted with caution, given the limited number of cargo-bearing regions and the predominantly draft status of most genomes. Furthermore, the A1 genome contained multiple restriction-modification systems, showed depletion of several methylation motif families in mobile regions, and had limited CRISPR spacer matching evidence, suggesting prior exposure to the relevant sequence space. None of the genomes met our predefined criteria for whole-genome convergence.Collectively, our results support a model in which S. parasuis accumulates AMR-related genes in a modular fashion via mobile elements within stable species boundaries, with no detectable same-MGE colocalization of AMR and virulence determinants under our analytical pipeline. These findings imply that AMR surveillance strategies for this species should prioritize tracking mobile genetic elements rather than inferring wholesale genomic convergence toward S. suis.
The study findings revealed that pELF-type plasmids utilize highly minimized conjugation machinery, which is similar to unusual systems previously identified in other gram-positive bacteria, such as Streptomyces.
Jun Kurushima, Natsuko Ota, Yuka Yoshii et al.· PLoS Pathogens· 0 citations
Background The rapid global spread of hypervirulence in Enterobacteriaceae, particularly in carbapenem-resistant Klebsiella pneumoniae, poses a significant public health threat. However, the key genetic vehicles and mechanisms driving horizontal transfer of hypervirulence-associated genes (iucA, iroB, rmpA, rmpA2, and peg-344) remain poorly defined, limiting effective surveillance. Methods We performed a large-scale genomic survey of 2,869 virulence-associated plasmid sequences and 2,337 complete Enterobacteriaceae chromosomes. Using comparative genomics and evolutionary analyses, we systematically identified and characterized Hypervirulence-associated Pseudo-Compound Transposons (Hva-PCTs), defined as structured mobile elements in which hypervirulence-associated genes are flanked by insertion sequences. Results Our results demonstrate that hypervirulence-associated genes are transmitted primarily as discrete IS-bounded units, which we term Hva-PCTs. We identified 29 distinct plasmid-borne Hva-PCTs (pHva-PCTs) and 30 chromosomal Hva-PCTs (cHva-PCTs). These modules show clear species-specific patterns: iucA/iroB-associated Hva-PCTs mainly originate in Escherichia coli and spread through IncFIB-containing multi-replicon plasmids (commonly combined with IncFIC(FII) and/or IncFII, while rmpA/rmpA2/peg-344-containing modules originate in K. pneumoniae and are disseminated via IncHI1B/repB plasmids. Three Hva-PCTs were detected on both plasmids and chromosomes (xHva-PCTs). In one clinical K. pneumoniae isolate (LS356), the identical composite module was present on both replicons. Simpler sub-modules, such as ISKqu3-rmpA2-iucA_1-IS102 and IS102-rmpA-peg-344-iroB_1-IS1A, frequently co-occur on the same plasmid; when positioned in tandem, they reconstitute the full composite structure. This assembly pattern is further supported by a partial duplication event in plasmid pP901. CD-HIT clustering (80% nucleotide identity and 90% coverage) showed that 13 of 22 major clusters contained both plasmid and chromosomal copies, with intra-cluster identities >80% across multiple sequence types and host species. Conclusion Hypervirulence-associated genes in Enterobacteriaceae are disseminated mainly as IS-flanked Hva-PCTs rather than solely through intact virulence plasmids. These modules exhibit strong but not absolute host specificity. The presence of identical Hva-PCTs on plasmids and chromosomes suggests inter-replicon mobility, while their stepwise assembly from simpler sub-modules highlights modular accretion as a key evolutionary process. Tracking Hva-PCTs as distinct mobile units may complement existing plasmid- and gene-centric surveillance approaches for hypervirulent and convergent strains. Experimental validation of their transposition activity and phenotypic effects is still required.
Shuaihua Fan, Lijun Wang, Chao Liu et al.· PLoS Pathogens· 0 citations
A systematic ESKAPE virome resource is established, phages’ dual roles in targeting resistant pathogens and curbing resistance spread are demonstrated, and mechanistic support for phage therapy clinical application is provided.
Pengwei Li, Quan Liu, Chunfang Deng et al.· bioRxiv· 0 citations
ABSTRACT The global rise of antimicrobial resistance (AMR) demands innovative strategies to limit the spread of multidrug-resistant bacteria. Conjugative plasmids, particularly those in the incompatibility group P (IncP), play a central role in disseminating resistance genes across bacterial species via their encoded type IV secretion system (T4SS). Here, we characterize the single-stranded RNA (ssRNA) bacteriophage (ssRNA phage) PRR1, which selectively targets bacteria carrying the IncP plasmid RP4, including many Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter species, and Escherichia coli (ESKAPEE) pathogens, and assess its ability to inhibit conjugation. Using cryo-electron microscopy, we resolved the mature PRR1 virion at 3.45 Å resolution, revealing two phage maturation protein (Mat)-RNA interactions within the 3′ untranslated region: a conserved interaction (Mat–U1) and a novel interaction (Mat–V1) for ssRNA phages. To characterize the PRR1–RP4 pilus interaction, we performed alanine-scanning mutagenesis and pinpointed four critical TrbC pilin residues (S12, W13, S72, and R77) for infection. Computational modeling revealed that these residues are located near the termini of the pilin at the phage–pilus interface. Notably, native and non-infectious, UV-cross-linked PRR1 was sufficient to block RP4 transfer, indicating conjugation inhibition does not require a complete infection cycle. Finally, combining PRR1 and antibiotic treatment yielded nine unique phage-resistant mutants within T4SS-associated genes on the RP4 plasmid. Eight of these mutants nearly abolished conjugation, while the trbE frameshift mutant retained ~30% of wild-type efficiency, which is pivotal to clarifying the relationship between phage infection and pilus function. Collectively, these results establish ssRNA phages as specific T4SS plasmid-targeting agents and underscore their potential to limit horizontal gene transfer in AMR pathogens. IMPORTANCE Antimicrobial resistance (AMR) spreads rapidly through horizontal gene transfer, largely driven by conjugative plasmids. Despite their central role, few strategies exist to directly block plasmid transfer. Here, we show that the IncP plasmid-dependent ssRNA phage PRR1 can inhibit the spread of antibiotic resistance genes by targeting the RP4 T4SS pilus. Structural and mutational analyses reveal previously unrecognized RNA packaging interactions and identify four pilin residues critical for infection. Remarkably, non-infectious PRR1 particles alone are sufficient to block conjugation, offering inhibition without the selective pressure from phage replication. Almost all PRR1-resistant RP4 mutants lost or had severely reduced plasmid transfer, while the remaining mutant is critical for studying the link between T4SS function and phage infection. These results highlight ssRNA phages as precise agents for limiting AMR gene dissemination. Antimicrobial resistance (AMR) spreads rapidly through horizontal gene transfer, largely driven by conjugative plasmids. Despite their central role, few strategies exist to directly block plasmid transfer. Here, we show that the IncP plasmid-dependent ssRNA phage PRR1 can inhibit the spread of antibiotic resistance genes by targeting the RP4 T4SS pilus. Structural and mutational analyses reveal previously unrecognized RNA packaging interactions and identify four pilin residues critical for infection. Remarkably, non-infectious PRR1 particles alone are sufficient to block conjugation, offering inhibition without the selective pressure from phage replication. Almost all PRR1-resistant RP4 mutants lost or had severely reduced plasmid transfer, while the remaining mutant is critical for studying the link between T4SS function and phage infection. These results highlight ssRNA phages as precise agents for limiting AMR gene dissemination.
Z. Lill, J. Thongchol, D. Solís et al.· Journal of Virology· 0 citations
Translucent post-larvae disease (TPD), caused by specific Vibrio parahaemolyticus strains, poses a global threat to shrimp aquaculture. A key virulence factor in these strains is the vhv gene cluster, which encodes toxins central to TPD pathogenesis. While plasmid-borne vhv genes have been extensively studied, the chromosome-borne vhv genes in TPD-associated strains remain poorly characterized. In this study, we systematically analyzed integrative mobilizable elements (IMEs) on the small secondary chromosomes of TPD-associated V. parahaemolyticus. Seven nearly identical novel IMEs harboring full-length vhv clusters were identified from seven independent strains. These IMEs encode a tyrosine recombinase for site-specific chromosomal integration, yet lack core conjugative transfer machineries including origin-of-transfer (oriT), relaxase, type IV coupling protein (T4CP), and IV secretion system (T4SS), consistent with the definition of non-autonomous IMEs. Each IME carries diverse functional cargo genes, covering five virulence factors (including previously unreported colonization and chemotaxis genes specific to TPD mobile elements), one pollutant-degrading enzyme, and two distinct anti-phage defense systems (PsyrTA and Lamassu-Fam), thereby potentially boosting the host’s fitness and pathogenicity in aquaculture environments. This study provides evidence for the critical role of IMEs in mediating the horizontal transfer of chromosomal vhv, offering new insights into TPD epidemiology and a foundation for developing targeted strategies to mitigate the disease’s impact on shrimp aquaculture.
Ruyi Hao, Yan Fang, Yang Yang et al.· Fishes· 0 citations
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