Introduction

Streptococcus suis (S. suis) is a gram-positive bacterial pathogen in the Streptococcus genus that causes substantial economic losses in the global pig industry1. The natural habitat of S. suis is the upper respiratory tract of pigs. It can also be found in the digestive tract and genitals2. S. suis causes outbreaks of meningitis, septicaemia, and pneumonia in neonatal piglets and adult pigs3,4. Moreover, S. suis is recognized as an emerging zoonotic pathogen that can cause meningitis, endocarditis, arthritis, sepsis, hearing loss, skin lesions and even death in humans5,6,7 . S. suis human infections have raised great public concern worldwide, and the majority of human infections occur in Thailand, Vietnam, and China7. S. suis sequence type (ST) 1 strains are the most frequently isolated from human cases worldwide, and ST1 has been predominant in sporadic human infections8,9. Multilocus sequence typing (MLST) analysis revealed that the ST 7 strains were derived from ST1 by the acquisition of 5 genomic islands10.

Most S. suis studies have been conducted on isolates from porcine sources11. However, studies on strains directly isolated from human CSF is rare. The genomic characteristics of S. suis strains directly isolated from human CSF remain poorly understood. Here, we sequenced the genome of an S. suis ST1 strain isolated from the CSF of a patient with severe meningitis. The findings of this study provided significant insight into the genomic characteristics, prediction of the pathogenic capacity, antimicrobial gene repertoire, putative virulence genes, and phylogenetic evolution of the isolate, enhancing the understanding of this zoonotic pathogen from human CSF.

Results

Antimicrobial susceptibility profiles

We identified resistance to erythromycin, clindamycin and tetracycline in the strain, along with susceptibility to penicillin, ampicillin, ceftriaxone, cefotaxime, cefepime, vancomycin, levofloxacin, linezolid, ertapenem, meropenem, and chloramphenicol (Table 1).

Genome composition analysis

The base quality scores for all positions were primarily concentrated in the green background area shown in Fig. 1, demonstrating high and consistent sequencing quality. A total of 15,350,328 reads were obtained after read cleaning. From these clean reads, the entire bacterial genome was successfully assembled into 34 contigs with a total length of 2,028,199 bp, a GC content of 41.21% and an N50 value of 131,426 bp (Table 2). The BUSCO (Benchmarking Universal Single-Copy Orthologues) assessment demonstrated high completeness in the genome assembly, with 96.6% of BUSCOs identified as complete, 2.6% as fragmented, and 0.9% as missing12. The genome assembly had an assembly depth of 200×, indicating that the genome was well assembled (Supplementary Table S1). The genome contains a total of 1,982 genomic features, including 1,933 coding genes and 49 RNA genes (3 5 S rRNA genes, 3 16 S rRNA genes, 1 23 S rRNA gene, 41 tRNA genes, and 1 ncRNA gene) (Supplementary Table S2). No plasmid was detected in the strain. MobileElementFinder13 detected 5 mobile genetic elements scattered across the genome, divided into 4 Insertion sequence(at contig 5, 6,14,21, respectively) and 1 Composite transposon(at contig6) (Supplementary Table S3). Analyses of the CRISPR–Cas system revealed one CRISPR array at contig 4 (CRISPR position 168638–168782, DR length = 43, number of spacers = 1), 1 CRISPR array at contig 16 (CRISPR: position 13657–13780, DR length = 32, number of spacers = 1) and two Cas clusters (CAS3_Typel). Based on clustering of allele genes (aroA, cpn60, dpr, gki, mutS, recA and thrA) with those of Streptococcus suis strains, in silico analysis of the assembled genome indicated that this strain was MLST sequence type 1 (ST1). The isolate was predicted to be pathogenic (probability of being a human pathogen = 0.921 out of 1.0, indicating greater pathogenicity). A circular genome map showing other genomic features is presented in Fig. 2.

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Quality scores across all bases.

Table 1 Antibiogram profiling of Streptococcus suis 366.
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Fig. 2
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Circular representation of the genome of highly pathogenic Streptococcus suis strain 366. The innermost circle represents the genomic sequence coordinates. Moving outwards, the circles represent GC content, GC skew (GC skewness, measuring the relative abundance of G and C, used to mark the starting and ending points on circular chromosomes), reverse-strand genes and forward-strand genes.

CARD annotation of Streptococcus suis 366

Two antimicrobial resistance genes, ermB and tet-(O), were detected in the genome using the Resistance Gene Identifier with the Comprehensive Antibiotic Resistance Database (CARD)14.Gene ermB confers resistance to erythromycin and clindamycin via antibiotic target alteration. Erm proteins dimethylate a single adenine in nascent 23 S rRNA, which is part of the large (50 S) ribosomal subunit15. As a consequence of methylation, binding of erythromycin to its target is impaired. The overlapping binding sites of macrolides and lincosamides in 23 S rRNA account for cross-resistance to the 2 classes of drugs16. Gene tet-(O) confers tetracycline resistance via antibiotic target protection. The tet(O) is a paralogue of the translational GTPase EF-G 6 and actively removes tetracycline from the ribosome in a GTP-hydrolysis-dependent fashion17. Both AMR genes were located at contig 9. No AMR genes were located on a mobile genetic element.

VFDB annotation results for Streptococcus suis 366

The genome of Streptococcus suis 366 harboured 4 virulence-associated genes, including hasC, cpsF, neuB, and pavA (Table 33). hasC, cpsF and neuB were related to capsule synthesis. There are more than 20 proposed virulence factors contributing to the pathogenesis of S.suis infections, including the capsular polysaccharide (CPS)18. However, the role in the pathogenesis of S. suis infections of most putative virulence factors (other than CPS) described so far remains to be confirmed19. The critical virulence factor described is the CPS20.CPS facilitates the survival of the strain in the bloodstream and the sialic acid component located in the terminal position of CPS is likely to be responsible for the antiphagocytosis18,21. These 4 virulence genes were located at separate contigs: hasC at contig 1, cpsF and neuB at contig 15, and pavA at contig 10. No virulence genes were located on mobile genetic elements.

Table 2 Assembly and annotation summary statistics.
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Table 3 Virulence genes in isolate Streptococcus suis 366.
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Gene Ontology (GO) annotation of Streptococcus suis 366

To elucidate the biological functions of the differentially expressed genes, GO enrichment analysis was performed22. A bar plot was constructed to visualize the enriched terms across the biological process, cellular component, and molecular function categories (Fig. 3). In the biological process category, terms related to ‘cellular process’ (Count = 282) and ‘metabolic process’ (Count = 261) were the most prominently enriched, suggesting that the gene set is primarily involved in cellular adaptation. For molecular function, genes were significantly enriched in ‘catalytic activity’ (Count = 204), suggesting that the genes predominantly encoded enzymes. With respect to cellular components, ‘protein-containing complex’ (Count = 67) was highly represented, indicating that the phenotype associated with the gene set is largely mediated by stable, multi-subunit macromolecular machines in which at least one component is a protein. The complete GO annotations are provided in Supplementary Table S4.

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Gene function classification of the Streptococcus suis 366 genome.

KEGG annotation of Streptococcus suis 366

The results of the alignment of the gene sets with the Kyoto Encyclopedia of Genes and Genomes (KEGG) database23 are shown in Fig. 4. KEGG pathway analysis revealed that the differentially expressed genes (DEGs) were significantly enriched in terms related to five major categories: cellular processes, environmental information processing, genetic information processing, metabolism and organismal systems. Several pathways were significantly enriched. The most prominent among these were ‘membrane transport’ (Count = 130), ‘carbohydrate metabolism’ (Count = 152), and ‘overview’ (Count = 115). In the membrane transport category, the most significantly enriched pathway was the ‘ko02010 ABC transporters pathway’, with 86 DEGs mapped to this pathway. The ABC (ATP binding cassette) transporter is among the active transport systems of cells and is known as the binding-lipoprotein-dependent transport system in gram-positive bacteria24. This pathway is known to play a pivotal role in environmental information processing and membrane transport. ABC transporters function as either importers, bringing nutrients and other molecules into cells, or exporters that pump toxins, drugs and lipids across membranes25,26. The complete KEGG pathway annotation is provided in Supplementary Table S5.

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KEGG pathway classification of the Streptococcus suis 366 genome.

Gene function annotation

Functional annotation was performed by alignment with the Cluster of Orthologous Groups of proteins (COG) database27 to analyse and infer gene functions. The COG annotation results for the isolate are shown in Fig. 5. The results indicated that the genome participated in various aspects of bacterial life processes. The functions of the genes in the Streptococcus suis 366 strain obtained in this study were mainly concentrated in categories such as E (amino acid transport and metabolism), G (carbohydrate transport and metabolism), J (translation, ribosomal structure, and biogenesis), and R (general function prediction), which are biologically significant for maintaining bacterial metabolism. Moreover, the functions of 147 genes were unknown and were concentrated in category S. The complete COG annotation is provided in Supplementary Table S6.

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COG annotation results for the Streptococcus suis 366 genome. Different colours represent distinct COG annotation categories. All COG annotation categories are labelled with letters, as shown on the right side of the figure.

Phylogenetic analysis based on 16 S rRNA

Phylogenetic analysis based on 16 S rRNA revealed that Streptococcus suis GZ1 (GCA 000018185.1) was the closest Streptococcus suis 366 relative18(Fig. 6). These strains were isolated from various countries, and the majority originated from China. Detailed information on these bacterial strains is provided in Supplementary Table S7.

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Phylogenetic relationship between Streptococcus suis 366 and the other Streptococcus suis strains based on 16 S rRNA. Different colours represent the different geographical origins of those strains.

Analysis of the pangenome

The results of the pangenome analysis of the S.suis 366 strain and its 7 closest relatives are presented in Fig. 7. Detailed information on related bacterial strains is provided in Supplementary Table S8. The pangenome of these strains comprised a total of 2300 genes. Among them, 1650 core genes were shared by the 8 strains. In addition, there are 413 shell genes and 237 cloud genes.

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Flower plot of homologous genes. The figure shows the numbers of common and unique orthologue clusters. The flower heart indicates the orthologue cluster in which all the strains are present, and the petals indicate those unique to each strain.

Phylogenetic analysis of Streptococcus suis 366 based on SNPs

The resolution power of the 16 S rRNA analysis is limited due to the predominance of conserved sites in sequences, not giving enough evolutionary information for the analysis and restricting the identification at the genus level28. Therefore, phylogenetic analysis based on SNPs was performed. Phylogenetic analysis based on SNPs revealed Streptococcus suis JS14 (GCA 0000186405.1) as the closest Streptococcus suis 366 relative (Fig. 8). Among the 8 strains, the ST1 type was dominant. ST1 and ST7 were closely related in terms of evolution. Some of the strains were derived from diseased pigs, and some were from humans. Compared to the 16 S rRNA single-gene phylogenetic tree, the genome-wide SNP-based phylogeny provides a more resolved and accurate representation of evolutionary relationships.

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Phylogenetic tree constructed based on the core-genome SNPs using the neighbour–joining method with the FastTree tool and visualized using the interactive Tree of Life tool. STSS: streptococcal toxic shock-like syndrome.

Comparative genomic analysis with the closest pathogenic strains

Multi-genome comparisons among S. suis strains GZ1, 366, and JS14 are shown in Fig. 9. The highly pathogenic strain ST1 GZ1 was isolated from a patient with septicemia in China. The genome of highly pathogenic strain ST1 GZ1 is a single circular chromosome of 2,038,034 bp with a GC content of 41.44% (CP000837). No plasmids were found and the chromosome has 1987 predicted coding sequences (CDSs)9. ST7 JS14 was isolated from articulation of a diseased pig in Jiangsu Province, China29. The genome of strain JS14 consists of a single circular chromosome which is 2,137,435 bp in length, with a GC content of 41.22%. No plasmids were found and the chromosome has 2,106 CDSs. Comparative analyses revealed that strain ST1 GZ1 acquired 1AMR tet(W)), JS14 gained 6 AMRs (APH(3’)-IIIa, tet(O), tet(40), SAT-4, aad(6), ErmB) and S.suis 366 harbored 2 AMRs (tet(O), ErmB).

Fig. 9
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Multi-genome comparisons among S. suis strains GZ1, 366, and JS14, performed using the Mauve software. Each color region refers to a locally collinear block (LCB). Colors are arbitrarily assigned to each LCB by the software. Vertical peaks in each LCB denote the variance of conservation. LCBs below the centerline of genomes are in reverse complement orientation.

Discussion and conclusion

Streptococcus suis, a zoonotic pathogen circulating in swine, can cause severe human infection and can lead to fatal complications, posing a serious public health threat worldwide30. MLST has been widely used to genetically classify bacterial strains. To date, ST1 has been predominantly reported to be responsible for human infections among invasive isolates worldwide7. However, few studies have investigated strains directly isolated from the cerebrospinal fluid of patients with purulent meningitis31.

The results indicated that the isolated strain 366 was highly resistant to erythromycin, clindamycin and tetracycline. CARD identified tet(O) and ermB, which confer resistance to tetracycline and macrolide-lincosamide in the strain. Previous studies have also revealed that tet(O) and ermB are commonly found in S. suis strains from pigs and humans worldwide32,33. Streptococcus suis 366 was predicted to be highly pathogenic (the probability of being a human pathogen = 0.921 out of 1.0). Notably, virulence-associated genes, such as mrp, sly, epf, ofs, revS, nadR, neuB, and neuC, are typically found in highly pathogenic S. suis strains18,34. In the study, three genes related to capsule synthesis, hasC, cpsF and neuB, were identified. The product is a capsule, which is undisputedly considered the major virulence factor35. S. suis survival in blood and its dissemination depend on the production of a thick capsule, which protects the bacterium against immune recognition and immune clearance36. Protein PavA was the first Fibronectin-binding protein identified in S. pneumoniae, which acts directly as a fibronectin adhesin37. The presence of these 4 genes may contribute to the virulence of the isolate by potentially enhancing its ability to evade the immune system and adhere to host.

Antimicrobial resistance genes spread rapidly due to different mobile genetic elements (MGEs)38. The primary MGE categories are insertion sequences (IS), transposons (Tn), gene cassette/integron systems (integrons), plasmids, integrative and conjugative elements (ICE), and bacteriophages39. The main driving force behind spreading resistance between the environmental and clinical bacteria is horizontal gene transfer mediated by MGEs, such as transposons, plasmids and lysogenic bacteriophages40. In this study, there are 5 MGEs scattering across the genome, divided into 4 IS and 1 Tn. 3 MGEs belong to ISL3 family and 2 MGEs belong to IS110 family. However, Comprehensive genomic analysis revealed that the identified virulence and antimicrobial resistance genes were not located on known mobile genetic elements. The absence of key virulence and resistance genes from mobile genetic elements suggests a lower immediate risk of horizontal gene transfer among bacterial populations. Instead, the dissemination of these strains is likely driven by clonal expansion, which has implications for outbreak management and source tracking.

Prokaryotes have evolved a wide repertoire of defence systems to prevent invasion by mobile genetic elements (MGEs). However, because MGEs are vehicles for the exchange of beneficial accessory genes, defence systems could consequently impede rapid adaptation in microbial populations41. CRISPR–Cas functions as a widespread adaptive immunity system that protects archaea and bacteria against viruses and other MGEs42. Analyses of the CRISPR–Cas system revealed one CRISPR array, 1 CRISPR array and two Cas clusters. The CRISPR structure may limit the horizontal transfer of drug resistance and virulence genes.

In this study, various databases and annotation tools were employed. Detailed analyses and interpretation of aspects such as the function and structure of each gene and their roles in the life activities of bacteria were carried out. The results of the GO analysis indicated that the genome was the most prominently enriched biological process category terms related to ‘cellular process’ and ‘metabolic process’, suggesting that the gene set is primarily involved in cellular adaptation. The most significantly enriched pathway was the ‘ko02010 ABC transporter pathway’, which may function as exporters that pump drugs across membranes. The results of COG analysis indicated that the functions of the genes were concentrated mainly in categories that were biologically significant for maintaining bacterial metabolism. Through these systematic and comprehensive gene annotations, the understanding of the Streptococcus suis ST1 genome has been further improved, laying a solid theoretical foundation for subsequent in-depth research on the biological characteristics and pathogenesis mechanisms of Streptococcus suis, as well as the development of targeted prevention and control strategies.

Phylogenetic analysis based on 16 S rRNA revealed that Streptococcus suis GZ1 is a close relative of the isolate characterized in this study. In a previous study, the highly pathogenic strain ST1 GZ1, which was isolated in 2005 from a patient in China who had septicemia, was representative of most strains isolated from humans in Europe and Asia9. Compared to Streptococcus suis GZ1, additional AMR gene ermB was found in Streptococcus suis 366. Furthermore, key virulence-associated factors (hasC, cpsF, neuB, and pavA) were identified in both strains. The SNP phylogeny provided a more resolved and accurate evolutionary relationship than the 16 S rRNA. The phylogenetic tree based on the core genome SNPs revealed Streptococcus suis JS14 as the closest Streptococcus suis 366 relative. S. suis strain ST7 JS14 was isolated from articulation of a diseased pig in Jiangsu Province, China29. A recent study demonstrated that epidemic ST7 strains evolved from ST1 strains via a single-nucleotide change in the housekeeping gene thyA9,10. Phylogenetic analysis in this study revealed that ST1 and ST7 are closely related and that there may be an evolutionary connection. Pangenome analysis revealed that ST1 and ST7 share a large number of core genes, indicating their evolution during evolution.

In previous study, a strain of S.suis serotype 2 was isolated from a pig farm and subjected to whole-genome sequencing and characterization. The study have identified potential virulence genes associated with S.suis serotype 2, laying the foundation for further research on the virulence and resistance of S.suis serotype 243. While our study employs the established methodological approach of genomic characterization with the previous published study, it is fundamentally distinguished by focusing on a clinically significant human ST1 isolate from purulent meningitis CSF. This direct analysis of a strain responsible for severe human disease provides unique insights into its pathogenesis. Furthermore, the inclusion of the strain from a new geographical region (Nanning) expands the diversity of sequenced isolates, offering a broader perspective on the pathogen’s genetic landscape. Ultimately, our work significantly advances the understanding of S.suis by elucidating the genomic characteristics of a human-derived ST1 strain and clarifying its evolutionary relationship with the ST7 lineage.

A study conducted an investigation into the phylogenetic structure, genomic features, and virulence levels of 73 S. suis ST1 human strains from Guangxi between 2005 and 2020, which revealed significant diversity in phylogenetic structure and virulence levels among ST1 Guangxi strains8. while both studies employ genomic analyses of S. suis ST1, our work provides distinct and complementary insights by characterizing a recently isolated (2025) clinical strain, thereby offering a contemporary genetic snapshot critical for tracking this evolving pathogen. Furthermore, by incorporating strains from multiple regions and diverse hosts, our pan-genome and phylogenetic analyses significantly enhance the representativeness of evolutionary inferences. Transcending a purely comparative approach, our study delivers a functional genomic profile through systematic annotation (GO, KEGG, COG), revealing key enrichments in cellular/metabolic processes and ABC transporters, which advances the understanding of its genetic repertoire. Notably, we uncovered a substantial shared core gene set and affirmed a close evolutionary relationship between ST1 and ST7. Collectively, these findings establish a crucial foundation for elucidating the biological characteristics of S. suis ST1, ultimately informing targeted control strategies.

This study has several limitations that warrant consideration. These limitations include reliance on a single isolate, lack of functional validation of genomic predictions, constraints of comparative datasets, and the challenges in inferring virulence or transmission pathways solely from genomic data. Consequently, caution is advised when extrapolating the results. Future research should prioritize multi-isolate comparisons, experimental validation of key genomic features, and the integration of complementary datasets to reinforce causal inference. Accordingly, our subsequent work will employ functional genetics assays, including gene knockout and complementation experiments, to empirically determine the mechanistic role of the CRISPR system in the horizontal transfer of antibiotic resistance and virulence genes.

In conclusion, this study provides the detailed genomic characteristics associated with ST1 in S.suis and enhances the understanding of this zoonotic pathogen isolated from human CSF. Moreover, the results enhance the understanding of the evolution connection of the ST1 and ST7 strains.

Methods

Patient with purulent meningitis and bacterial isolation

The clinical strain Streptococcus suis 366 was isolated from the cerebrospinal fluid of a patient with purulent meningitis in the emergency intensive care unit. The patient demonstrated partial improvement in cognitive function following targeted antimicrobial therapy. However, severe sensorineural hearing loss was observed. The cultured isolate was identified by a matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) system (with the IVD database version 3.0, Bruker, Germany).

Antimicrobial susceptibility testing

Antimicrobial susceptibility was determined using the BD Phoenix™ M50 Automated Microbiology System (Becton, Dickinson and Company, USA). Ten types of commonly used drugs were selected, namely, aminoglycosides, cephems, glycopeptides, fluoroquinolones, oxazolidinones, carbapenems, phenicols, macrolides, lincosamides, and tetracyclines. Streptococcus pneumoniae ATCC 49619 was used as a quality control strain. The broth microdilution method was carried out in strict accordance with the current Clinical and Laboratory Standards Institute (CLSI) performance standards (M100-Ed35, 2025)44, and the breakpoints for β-haemolytic streptococci were used.

DNA extraction and whole-genome sequencing

The bacterial genomic DNA was extracted using a bacterial genomic DNA isolation kit (Sangon Biotech, Shanghai, China). Whole-genome sequencing was subsequently conducted on the Illumina NovaSeq 6000 platform. Raw sequencing reads were processed through quality control analysis using FastQC45, followed by adapter trimming and quality filtering with Trimmomatic46. De novo genome assembly was ultimately performed using the SPAdes47 genome assembler (v3.5.0) with default parameters, and the assembled genome was annotated using the NCBI Prokaryote Genome Annotation Pipeline48. Busco V6.0.0 was used to assess the assembled bacterial genome12.

Genome annotation

Multilocus sequence typing (MLST) was performed with MLST 2.0 using default parameters to determine the strain lineage49. CRISPR–Cas systems were annotated using CRISPRCasFinder to assess adaptive immunity elements50. The presence of plasmids was analysed using PlasmidFinder 2.151. Circular Streptococcus suis 366 genome visualization was performed using Prokesee52. The mobile genetic elements in the genome were detected using MobileElementFinder13. The computational screening of antimicrobial resistance (AMR) genes was conducted through the Comprehensive Antibiotic Resistance Database using rigorous identity and coverage thresholds (≥ 90%)53. Virulence-associated genes were identified by alignment against the Virulence Factor Database (VFDB)54, with a sequence identity threshold of 70%. Pathogenic potential was predicted using PathogenFinder, which evaluates host-specific pathogenicity based on genomic signatures55. Functional annotations, including Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and Clusters of Orthologous Groups of proteins (COG) analyses, were conducted for Streptococcus suis 36656,57,58.

16 S rRNA-based phylogenetic analysis

The 16 S rRNA sequence of Streptococcus suis 366 was aligned to the NCBI database using BLAST with the parameter identity > 95%. Afterwards, the selection criteria for 16 S rRNA sequences included the following: (1) belonging to sequence type 1 (ST1); (2) being isolated from cerebrospinal fluid; (3) being isolated from infected pigs or human. Then, a phylogenetic tree was constructed using FastTree59 after multiple sequence alignment and clipping using MAFFT60.

Pangenome construction

The Roary program61 was used to construct the pangenome with a minimum percentage identity of 95%. The common genes and unique genes of Streptococcus suis 366 and 7 related strains were obtained and analysed in depth. Functional annotation was performed by alignment with the Cluster of Orthologous Groups of proteins (COG) database to analyse and infer gene functions.

SNP-based phylogenetic analysis

Based on the results of whole-genome SNP analyses, a neighbour-joining phylogenetic tree was constructed with FastTree59 incorporating 7 closely related reference strains (≥ 95% average nucleotide identity). The resultant phylogenetic topology was subsequently visualized using the Interactive Tree of Life (iTOL) web platform62.

Genome comparisons with the closest pathogenic strains

In addition, genome comparisons were undertaken between the three genomes of the ST1 highly virulent strain S. suis GZ1, ST7 epidemic S. suis JS14, and the study strain S. suis 366, using the Mauve software and following the instructions63.