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The Streptococcus suis (S. suis) is an important zoonotic pathogen that causes streptococcal disease in pigs and poses a threat to humans. This study provides an understanding of the prevalence of  S.suis in eastern China and provides guidance for clinical prophylaxis. From 2021 to 2023, a total of 143 strains of S. suis were isolated from 1642 lung tissue and nasal swabs from healthy and suspected infected pigs in Shandong Province, China, using the Phenotypic tests and PCR technique. The isolates were then tested for serotype, virulence-related genes, and resistance genes. Among the 143 isolates, type 2 was the predominant serotype with 98 isolates (98/143, 68.5%), followed by type 5 with 22 isolates (22/143, 15.3%), type 4 with 6 isolates (6/143, 4.2%), type 19 with 4 isolates (4/143, 2.8%) and type 21 with 5 isolates (5/143, 3.5%), respectively. A minimum of 78.3% of the strains exhibited the presence of virulence-related genes including pgda, dlta, mann, fbps, orf2, and sspa, whereas the virulence-associated genes Sum, Sly, and Salkr are not widely prevalent. For the detection of resistance genes, it was found that the tetO gene had a high detection rate of 70.1% (101/143), whereas neither the pbp2b gene nor the cat1 and cat2 genes were detected. Antimicrobial susceptibility testing revealed that 96.5% (138/143) of the isolates exhibited multidrug resistance (MDR). And polypeptide B was found to be tolerated by 125 of the 143 strains (87.4%). Although we did not detect the β-lactam resistance gene in any of the 143 strains, an average of 39.2% of the strains were resistant to β-lactam antibiotics. The results of the current study is thought it may be help to understand the prevalence of S. suis and provide important insights into treatment and prevention., Competing Interests: Declarations. Competing interests: The authors declare no competing interests. Ethical approval: Ethical approval for these studies was obtained from the Ethics Committee of Shandong First Medical University & Shandong Academy of Medical Sciences under approval number LS2024015. The animals’ owners were informed and consented to the above study. The collection of samples was conducted without the administration of anesthesia or euthanasia of the animals, and lung tissue was obtained from pigs afflicted with the disease. All animal experiments comply with China’s Regulations on the Administration of Laboratory Animals. All animal experiments has been reported in accordance with ARRIVE guidelines., (© 2025. The Author(s).)

Streptococcus suis (S. suis) is a major pathogen for pigs, causing large economic losses to the swine industry. Moreover, this bacterium has a zoonotic potential, being capable of infecting humans in close contact with pigs or, less frequently, through contact with pork products. Given its importance in both veterinary and public health, S. suis remains a key topic of research. This study explores the genetic characteristics of 154 S. suis isolates obtained from clinical samples collected from pigs between 2018 and 2022. Whole genome sequencing (WGS) allowed a comprehensive analysis of the S. suis population in Spain, including detection of serotype, sequence type (ST), antimicrobial resistance genes, and virulence-associated genes. This approach also explored the vertical transmission of this pathogen through vertically integrated pyramids, as evidenced by associations between grandmother and mother sow farms, and phylogenetic groups, serotypes, and STs. Our analysis revealed that serotype 9 was the most prevalent in our strain collection, predominantly associated with ST123. Notably, the three most significant virulence genes, encoding the extracellular protein factor (EPF), the muramidase-release protein (MRP), and suilysin (SLY), were not consistently present in all clinical isolates. Regarding antimicrobial resistance, no phenotypic resistance was observed to ceftiofur or florfenicol, while observing low resistance to ampicillin (0.6 %) and enrofloxacin (2.6 %), intermediate resistance to penicillin (22.1 %), and high percentage of non-wild-type isolates to trimethoprim-sulfamethoxazole (57.1 %), and doxycycline (96.1 %). The most prevalent antimicrobial resistance genes (ARGs) were tet(O) (85.1 %) and erm(B) (86.4 %), conferring resistance to tetracyclines and macrolides, respectively, although macrolides were not included in the phenotypic testing. Overall, this study provides key epidemiological insights into this significant systemic pathogen within the Spanish swine population. The findings underscore the importance of understanding sample origins, such as grandmother and mother sow farms, to develop an effective antimicrobial stewardship program for managing S. suis-associated diseases., Competing Interests: Declaration of Competing Interest 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 © 2025 Elsevier B.V. All rights reserved.)

Streptococcus suis serotype 2 (SS2) is an emerging zoonotic agent responsible for a variety of diseases. The septicemia caused by SS2 suggests that it can evade the bactericidal effects of innate immune cells. However, the mechanisms by which SS2 evades innate immunity remain largely unknown. Neutrophils are critical components of innate immunity for antimicrobial defense. In this study, we identified a cell surface protein (CSP) insertion mutant in an SS2 transposon mutant library that significantly induces neutrophil extracellular traps (NETs) and shows poor survival compared to the parent SS2 in the bloodstream. CSP deletion mutant (ΔCSP) was constructed and it exhibits a defective polysaccharide capsule and enhanced biofilm formation. Although ΔCSP induces increased NET release and ROS formation, its survival and proliferation within neutrophils and NETs are significantly reduced. Additionally, ΔCSP stimulates extracellular signal-regulated kinase 1/2 (ERK1/2) signaling in neutrophils, which may enhance neutrophil bactericidal activity. Importantly, purified polysaccharide capsule from SS2 can inhibit neutrophil bactericidal effects. This study identifies bacterial virulence factors that prevent SS2 clearance by neutrophils, offering potential antigens or drug targets for the prevention and control of swine streptococcosis., 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 © 2025 Elsevier Ltd. All rights reserved.)

Streptococcus suis (S. suis) is a major pathogen in swine and poses a potential zoonotic threat, which may cause serious diseases. Many toxin-antitoxin (TA) systems have been discovered in S. suis, but their functions have not yet been fully elucidated. In this study, an auto-regulating type II TA system, ParDE, was identified in S. suis serotype 2 strain ZY05719. We constructed a mutant strain, ΔparDE, to explore its functions in bacterial virulence, various stress responses, and biofilm formation capabilities. The toxicity exerted by the toxin ParE can be neutralized by the antitoxin ParD. The β-galactosidase activity analysis indicated that ParDE has an autoregulatory function. An electrophoretic mobility shift assay (EMSA) confirmed that the antitoxin ParD bound to the promoter of ParDE as dimers. In the mouse infection model, the deletion of ParDE in ZY05719 significantly attenuated virulence. ΔparDE also exhibited a reduced anti-oxidative stress ability, and ΔparDE was more susceptible to phagocytosis and killing by macrophages. Moreover, the biofilm formation ability of the ΔparDE strain was significantly enhanced compared to ZY05719. Taken together, these findings indicate that the type II TA system ParDE plays a significant role in the pathogenesis of S. suis, providing new insights into its pathogenic mechanisms., Competing Interests: Declarations. Ethics approval and consent to participate: All experimental protocols were approved by the Animal Ethics Committee of Nanjing Agricultural University. All methods were carried out in strict accordance with the animal welfare standards of the Guidelines of the Jiangsu Provincial Animal Research Committee (License Number: SYXK(SU) 2021-0086). Consent for publication: Not applicable. Competing interests: The authors declare no conflicts of interest with the contents of this article., (© 2025. The Author(s).)

Streptococcus suis is a causative agent of swine and human infections. Genomic analysis indicated that eight S. suis serotype 5 strains recovered from human patients and pigs carried many virulence-associated genes and markers defining pathogenic pathotypes. The strains were sequence types diverse and clustered within either minimum core genome group 3 (MCG-3) or MCG-7-3. Almost all the serotype 5 strains were non-susceptible to penicillin, ceftriaxone, erythromycin, and levofloxacin. Resistance to tetracycline and clindamycin was observed in all strains. The antimicrobial resistance genes tet(O), tet(O/W/32/O), tet(W), tet(44), erm(B), ant(6)-Ia, lsaE, and lnuB were found in these strains. Moderate-to-large numbers of substitutions were observed in three penicillin-binding proteins (PBP)-PBP1A, PBP2B, and PBP2X-in the penicillin-non-susceptible serotype 5 isolates that were involved in β-lactam-non-susceptibility. Comparative genomics between the serotype 5 and 2 strains revealed that only 15 genes absent from the serotype 2 strains were shared by all the serotype 5 strains. However, some additional genes were present only in some of the serotype 5 strains. This study highlighted the pathogenic potential of virulent serotype 5 strains in humans and pigs and the need for increased monitoring of penicillin-non-susceptibility in S. suis serotypes other than for serotype 2., Competing Interests: Declaration of competing interest The authors report there are no competing interests to declare., (Copyright © 2023 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Bacterial DeoR family transcription regulators regulate multiple physiological processes. Little is known about the function of DeoR family regulators in streptococci. Here, we identified a novel DeoR family regulator, GlpR, from Streptococcus suis, a pathogen causing severe diseases in pigs and humans. GlpR was involved in glycerol utilization and exhibited specific signature residues at positions 30-31 (KV) which are crucial for DNA binding. Deletion of glpR (ΔglpR) showed a significant increase in relative growth rate in glycerol medium compared to the wild-type (WT) and complementary strains (CΔglpR). Employing RNA-seq analysis, β-galactosidase activity analysis, and electrophoretic mobility shift assay, we discovered that GlpR directly represses the expression of glycerol metabolism-related genes pflB2, pflA1, and fsaA, encoding pyruvate formate-lyase and its activating enzyme, and fructose-6-phosphate aldolase, respectively. Compared to WT and CΔglpR, ΔglpR showed a reduced survival rate under oxidative stress and in murine macrophages and attenuated virulence in mice. GlpR probably enhances oxidative stress resistance and virulence in S. suis by functioning as a glycerol metabolic repressor decreasing energy consumption. These findings contribute to a better understanding of S. suis pathogenesis and enrich our knowledge of the biological functions of DeoR family regulators in streptococci., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest., (Copyright © 2024 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Zoonotic streptococci cause several invasive diseases with high mortality rates, especially meningitis. Numerous studies elucidated the meningitis pathogenesis of zoonotic streptococci, some specific to certain bacterial species. In contrast, others are shared among different bacterial species, involving colonization and invasion of mucosal barriers, survival in the bloodstream, breaching the blood-brain and/or blood-cerebrospinal fluid barrier to access the central nervous system, and triggering inflammation of the meninges. This review focuses on the recent advancements in comprehending the molecular and cellular events of five major zoonotic streptococci responsible for causing meningitis in humans or animals, including Streptococcus agalactiae, Streptococcus equi subspecies zooepidemicus, Streptococcus suis, Streptococcus dysgalactiae, and Streptococcus iniae. The underlying mechanism was summarized into four themes, including 1) bacterial survival in blood, 2) brain microvascular endothelial cell adhesion and invasion, 3) penetration of the blood-brain barrier, and 4) activation of the immune system and inflammatory reaction within the brain. This review may contribute to developing therapeutics to prevent or mitigate injury of streptococcal meningitis and improve risk stratification., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest. The figures were created with BioRender software., (Copyright © 2024 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Streptococcus suis is a significant and emerging zoonotic pathogen. ST1 and ST7 strains are the primary agents responsible for S. suis human infections in China, including the Guangxi Zhuang Autonomous Region (GX). To enhance our understanding of S. suis ST1 population characteristics, we conducted an investigation into the phylogenetic structure, genomic features, and virulence levels of 73 S. suis ST1 human strains from GX between 2005 and 2020. The ST1 GX strains were categorized into three lineages in phylogenetic analysis. Sub-lineage 3-1a exhibited a closer phylogenetic relationship with the ST7 epidemic strain SC84. The strains from lineage 3 predominantly harboured 89K-like pathogenicity islands (PAIs) which were categorized into four clades based on sequence alignment. The acquirement of 89K-like PAIs increased the antibiotic resistance and pathogenicity of corresponding transconjugants. We observed significant diversity in virulence levels among the 37 representative ST1 GX strains, that were classified as follows: epidemic (E)/highly virulent (HV) (32.4%, 12/37), virulent plus (V+) (29.7%, 11/37), virulent (V) (18.9%, 7/37), and lowly virulent (LV) (18.9%, 7/37) strains based on survival curves and mortality rates at different time points in C57BL/6 mice following infection. The E/HV strains were characterized by the overproduction of tumour necrosis factor (TNF)-α in serum and promptly established infection at the early phase of infection. Our research offers novel insights into the population structure, evolution, genomic features, and pathogenicity of ST1 strains. Our data also indicates the importance of establishing a scheme for characterizing and subtyping the virulence levels of S. suis strains.

The arginine deiminase system (ADS) has been identified in various bacteria and functions to supplement energy production and enhance biological adaptability. The current understanding of the regulatory mechanism of ADS and its effect on bacterial pathogenesis is still limited. Here, we found that the XRE family transcriptional regulator XtrSs negatively affected Streptococcus suis virulence and significantly repressed ADS transcription when the bacteria were incubated in blood. Electrophoretic mobility shift (EMSA) and lacZ fusion assays further showed that XtrSs directly bind to the promoter of ArgR, an acknowledged positive regulator of bacterial ADS, to repress ArgR transcription. Moreover, we provided compelling evidence that S. suis could utilize arginine via ADS to adapt to acid stress, while Δ xtrSs enhanced this acid resistance by upregulating the ADS operon. Moreover, whole ADS-knockout S. suis increased arginine and antimicrobial NO in the infected macrophage cells, decreased intracellular survival, and even caused significant attenuation of bacterial virulence in a mouse infection model, while Δ xtrSs consistently presented the opposite results. Our experiments identified a novel ADS regulatory mechanism in S. suis , whereby XtrSs regulated ADS to modulate NO content in macrophages, promoting S. suis intracellular survival. Meanwhile, our findings provide a new perspective on how Streptococci evade the host's innate immune system.

Streptococcus suis is a major bacterial pathogen in pigs and an emerging zoonotic pathogen. Different S. suis serotypes exhibit diverse characteristics in population structure and pathogenicity. Surveillance data highlight the significance of S. suis serotype 4 (SS4) in swine streptococcusis, a pathotype causing human infections. However, except for a few epidemiologic studies, the information on SS4 remains limited. In this study, we investigated the population structure, pathogenicity, and antimicrobial characteristics of SS4 based on 126 isolates, including one from a patient with septicemia. We discovered significant diversities within this population, clustering into six minimum core genome (MCG) groups (1, 2, 3, 4, 7-2, and 7-3) and five lineages. Two main clonal complexes (CCs), CC17 and CC94, belong to MCG groups 1 and 3, respectively. Numerous important putative virulence-associated genes are present in these two MCG groups, and 35.00% (7/20) of pig isolates from CC17, CC94, and CC839 (also belonging to MCG group 3) were highly virulent (mortality rate ≥ 80%) in zebrafish and mice, similar to the human isolate ID36054. Cytotoxicity assays showed that the human and pig isolates of SS4 strains exhibit significant cytotoxicity to human cells. Antimicrobial susceptibility testing showed that 95.83% of strains isolated from our labs were classified as multidrug-resistant. Prophages were identified as the primary vehicle for antibiotic resistance genes. Our study demonstrates the public health threat posed by SS4, expanding the understanding of SS4 population structure and pathogenicity characteristics and providing valuable information for its surveillance and prevention.

Streptococcus suis ( S. suis ) is an important swine bacterial pathogen and causes human infections, leading to a wide range of diseases. However, the role of 5'-nucleotidases in its virulence remains to be fully elucidated. Herein, we identified four cell wall-anchored 5'-nucleotidases (Snts) within S. suis , named SntA, SntB, SntC, and SntD, each displaying similar domains yet exhibiting low sequence homology. The malachite green reagent and HPLC assays demonstrated that these recombinant enzymes are capable of hydrolysing ATP, ADP, and AMP into adenosine (Ado), with the hierarchy of catalytic efficiency being SntC>SntB>SntA>SntD. Moreover, comprehensive enzymatic activity assays illustrated slight variances in substrate specificity, pH tolerance, and metal ion requirements, yet highlighted a conserved substrate-binding pocket, His-Asp catalytic dyad, metal, and phosphate-binding sites across Snts, with the exception of SntA. Through bactericidal assays and murine infection assays involving in site-mutagenesis strains, it was demonstrated that SntB and SntC collaboratively enhance bacterial survivability within whole blood and polymorphonuclear leukocytes (PMNs) via the Ado-A2aR pathway in vitro , and within murine blood and organs in vivo . This suggests a direct correlation between enzymatic activity and enhancement of bacterial survival and virulence. Collectively, S. suis 5'-nucleotidases additively contribute to the generation of adenosine, influencing susceptibility within blood and PMNs, and enhancing survival within blood and organs in vivo . This elucidation of their integral functions in the pathogenic process of S. suis not only enhances our comprehension of bacterial virulence mechanisms, but also illuminates new avenues for therapeutic intervention aimed at curbing S. suis infections.

As a zoonotic pathogen, S. suis serotype 2 (SS2) can cause severe diseases in both pigs and humans, and develop resistance to antibiotics. Plant natural compounds are regarded as promising alternatives to conventional antibiotics. Phillyrin is the major bioactive components of Chinese herbal medicine Forsythia suspensa. In this study, we explored the activity and action mechanism of phillyrin against SS2. The results showed that phillyrin could disrupt membrane integrity, destroy intracellular structures, and increase the exosmosis of DNA. Results of PCR revealed that phillyrin affected bacterial-virulence-related genes' expression levels. Meanwhile, phillyrin significantly decreased the adhesion activity, inhibited lactate dehydrogenase (LDH) secretion, and reduced biofilm formation of SS2 in Newborn pig trachea epithelial (NPTr) cells. Furthermore, phillyrin protected tight junction protein of NPTr cells from SS2. We reported that phillyrin (0.1 mg/kg) treatment after bacterial challenge significantly improved the survival rate, ameliorated pulmonary inflammation, and inhibited the accumulation of multiple cytokines (IL-1, IL-6, IL-8, and TNF-α). Molecular docking showed that phillyrin had a good binding activity with the Ala88 and Asp111 of suilysin (SLY), one of the most important virulence factors of SS2. Collectively, phillyrin possesses antibacterial and anti-inflammatory activities, and is a promising candidate for preventing SS2 infection.

Streptococcus suis (S. suis) represents a significant bacterial pathogen, with its zoonotic transmission from infected or deceased pigs to humans posing a serious threat to public health. The type IV secretion system (T4SS), a critical virulence factor of S. suis, is tightly regulated by diverse environmental conditions. This study explores the influence of environmental variables, including temperature, incubation duration, monosaccharides, and metal ions, on the regulation of T4SS in S. suis and its associated pathogenicity. Results revealed that T4SS expression peaked during the stabilization phase at 37 °C, with galactose markedly enhancing T4SS expression relative to glucose. Zinc ions specifically enhanced the expression of the T4SS effector SspA among various metal ions. Moreover, zinc exposure significantly augmented both T4SS and virulence gene expression capabilities in S. suis. Zinc-treated S. suis exhibited enhanced adhesion, invasion, and colonization capacity in Hep-2 cells, Raw264.7 cells, and mouse models. These findings provide a deeper comprehension of the environmental modulation of T4SS in S. suis, paving the way for advanced studies into its mechanisms of pathogenicity., 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 © 2025 Elsevier Inc. All rights reserved.)

Streptococcus suis negatively impacts swine health, posing diagnostic and preventative challenges. S. suis can induce disease and also quietly reside on mucosal surfaces. The limited use of diagnostic tools to identify disease-associated strains and rule out differential diagnoses, alongside the complex ecology of S. suis , poses significant challenges in comprehending this important pathogen and defining pathotypes. This study evaluated 2,379 S . suis central nervous system (CNS) isolates from diagnostic submissions between 2015 and 2019. Isolates originating from submissions with histologic evidence of CNS infection ( n = 1,032) were further characterized by standard and advanced diagnostic techniques. We identified 29 S . suis serotypes and 4 reclassified serotypes as putative causes of CNS disease. Among these, serotypes 1 and 7 emerged as the predominant putative causes of CNS infection (32% of submissions). Furthermore, 51 sequence types (STs), of which 15 were novel, were detected with ST1 predominating. Through whole-genome sequencing of 145 isolates, we observed that five commonly used virulence-associated genes (VAGs; epf , mrp , sly , ofs , and srtF ) were not present in most disease-associated isolates, and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) yielded false-positive results in 7% of isolates. These data indicate that (i) clinical signs and site of isolation alone are insufficient for defining a pathotype, (ii) S. suis serotypes and STs associated with CNS infection are more diverse than previously reported, (iii) MALDI-TOF MS may need to be supplemented with additional diagnostic tools for precise S. suis identification, and (iv) VAGs remain an unreliable means for identifying isolates associated with CNS disease.IMPORTANCE Streptococcus suis is an important and complex systemic bacterial pathogen of swine. Characterization of S. suis strains originating from pigs with histologic confirmation of neurologic disease is limited. Review of swine diagnostic submissions revealed that fewer than half of cases from which S. suis was isolated from the brain had histologic evidence of neurologic disease. This finding demonstrates that clinical signs and site of isolation alone are not sufficient for identifying a neurologic disease-associated strain. Characterization of strains originating from cases with evidence of disease using classic and advanced diagnostic techniques revealed that neurologic disease-associated strains are diverse and commonly lack genes previously associated with virulence., Competing Interests: The authors declare no conflict of interest.

The thioredoxin (Trx) system plays a vital role in protecting against oxidative stress and ensures correct disulfide bonding to maintain protein function. Our previous research demonstrated that TrxA of Streptococcus suis Serotype 2 (SS2), a clinical strain from the lung of a diseased pig, contributes to virulence but is not involved in antioxidative stress. In this study, we identified another gene in the Trx family, TrxC, which encodes a protein of 104 amino acids with a CGDC active motif and 22.4 % amino acid sequence homology with TrxA. Unlike the TrxA, TrxC mutant strains were more susceptible to oxidative stresses induced by hydrogen peroxide and paraquat. In vitro experiments, the survival rate of the TrxC deletion mutant in RAW264.7 macrophages was only one-eighth of that of TrxA mutant strains. Transcriptome analysis revealed that autophagy-related genes were significantly upregulated in the TrxC mutant compared to those in the wild-type or TrxA mutant strains. Co-localization of LC3 puncta with TrxC was confirmed using laser confocal microscopy, and autophagy-related indicators were quantified using western blotting. Autophagy deficiency induced by ATG5 knockout significantly increased SS2 survival rate, especially in TrxC mutant strains. For the upstream signal regulation pathways, we found ΔTrxC strains regulate autophagy by activation of PI3K/Akt/mTOR signaling in RAW264.7 macrophages. In the Akt1-overexpressing cell line, ΔTrxC infection significantly decreased the autophagic response and promoted ΔTrxC mutant strain survival, while inhibition of Akt with MK2206 resulted in reduced ΔTrxC mutant strain survival and enhance the autophagic response. Furthermore, loss of TrxC increased the activity of MSR1, thereby inducing cellular autophagy and phagocytosis. Our data demonstrate that TrxC of SS2 contributes to virulence by inducing antioxidative stress and inhibits autophagy via the PI3K-Akt-mTOR pathway in macrophages, with MSR1 acting as a key factor in controlling infection., Competing Interests: Declaration of Competing Interest The authors declare they have no competing interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Streptococcus suis (S. suis) causes serious diseases in pigs, and certain serotypes also pose a risk to humans. The expression of capsular polysaccharides (CPS) is considered an important virulence property of the pathogen. Recently, some serotypes have been reclassified as other organisms, while novel S. suis serotypes are being described. Although the CPS can be typed by serological methods using antisera, the presence of unique sequences for each capsular polysaccharide synthesis locus (cps locus) enables convenient PCR-based serotyping. In this study, we characterized 33 non-serotypeable S. suis strains obtained from diseased pigs in the Czech Republic by sequencing and analyzing the cps locus. Phylogenetic analysis of cpn60 confirmed that all isolates belong to the S. suis species. Four isolates had cps loci similar to the previously described reference S. suis serotypes. Eleven isolates were classified as recently described novel cps loci (NCLs). Nine isolates had substitutions, insertions and/or deletions in their cps loci and showed only partial similarity to the already described NCLs. Another eight isolates had previously undescribed cps locus structures and were proposed as novel NCLs. One isolate had lost the genes encoding capsule biosynthesis. Only four sequence types (ST) had two isolates each; the rest had unique STs. Two isolates harbored the classical virulence associated genes (VAGs) mrp and sly. Another isolate had only the mrp gene, while a different isolate harbored only the sly gene. This study provides insight into untypeable isolates in the Czech Republic, highlighting the genetic diversity and potential for novel serotype identification., 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.)

Streptococcus suis is the most common and severe cause of meningitis in Vietnam, but the recent data about epidemiological characteristics and sequelae are still inadequate. We performed a retrospective study to describe the epidemiology, clinical characteristics, and outcomes of patients with S. suis-associated meningitis. A retrospective study was carried out by searching records from the Hospital for Tropical Diseases, Ho Chi Minh City, Vietnam, between January 2016 and January 2020. All data concerning epidemiological, clinical, and subclinical characteristics and treatment results were collected and analyzed. Over the study period, 153 cases were included in this study, with 95 cases occurring in the 2016-2017 period. The median age of all patients was 52 years (range, 29-90 years), and 70.6% of patients were male. Fifty-two patients (34%) were farmers, and the Mekong Delta was the region with the highest number of cases (60.1%). The most frequent manifestation of infection was fever (151/153, 98.7%), followed by hearing loss (64/153, 41.8%), and one patient (0.7%) died. All isolates remained susceptible to ceftriaxone, penicillin, vancomycin, and levofloxacin, whereas 99.2% of isolates were resistant to tetracycline. Occupational exposure to pigs and the consumption of contaminated food have been identified as primary risk factors associated with this zoonosis, and our study found no significant changes in epidemiological factors compared with past data. The Mekong Delta continues to be the most affected region for S. suis meningitis in southern Vietnam, with S. suis serotype 2 remaining the most relevant agent.

The complement system is the first defense line of the immune system. However, pathogens have evolved numerous strategies to evade complement attacks. Streptococcus suis is an important zoonotic bacterium, harmful to both the pig industry and human health. ApuA has been reported as a bifunctional amylopullulanase and also contributed to virulence of S. suis. Herein, we found that ApuA could activate both classical and alternative pathways of the complement system. Furthermore, by using bacterial two-hybrid, far-western blot and ELISA assays, it was confirmed that ApuA could interact with complement C3b. The interaction domain of ApuA with C3b was found to be its α-Amylase domain (ApuA_N). After construction of an apuA mutant (ΔapuA) and its complementary strain, it was found that compared to the wild-type strain (WT), ΔapuA had significantly increased C3b deposition and membrane attack complex formation. Additionally, ΔapuA showed significantly lower survival rates in human serum and blood and was more susceptible to engulfment by neutrophils and macrophages. Mice infected with ΔapuA had significantly higher survival rates and lower bacterial loads in their blood, lung and brains, compared to those infected with WT. In summary, this study identified ApuA as a novel factor involved in the complement evasion of S. suis and suggested its multifunctional role in the pathogenesis of S. suis., Competing Interests: Declaration of Competing Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Mobile genetic elements (MGEs) enable bacteria to acquire novel genes and traits. However, the functions of cargo genes within MGEs remain poorly understood. The cadmium resistance operon cadDX is present in many gram-positive bacteria. Although cadDX has been reported to be involved in metal detoxification, its regulatory mechanisms and functions in bacterial pathogenesis are poorly understood. This study revealed that cadDX contributes to cadmium resistance, oxidative stress resistance, and virulence in Streptococcus suis, an important zoonotic pathogen in pigs and humans. CadX represses cadD expression by binding to the cadDX promoter. Notably, cadX responds to H 2 O 2 stress through an additional promoter within the cadDX operon, mitigating the harmful effect of excessive cadD expression during oxidative stress. cadDX resides within an 11 K integrative and mobilizable element that can autonomously form circular structures. Moreover, cadDX is found in diverse MGEs, accounting for its widespread distribution across various bacteria, especially among pathogenic streptococci. Transferring cadDX into another zoonotic pathogen, Streptococcus agalactiae, results in similar phenotypes, including resistance to cadmium and oxidative stresses and increased virulence of S. agalactiae in mice. The new functions and regulatory mechanisms of cadDX shed light on the importance of the cadDX system in driving evolutionary adaptations and survival strategies across diverse gram-positive bacteria., (© 2024. The Author(s).)

Streptococcus suis (S. suis) disease is a prevalent zoonotic infectious threat that elicits a systemic inflammatory response in both swine and humans, frequently culminating in high mortality rates. The excessive inflammation triggered by S. suis infection can precipitate tissue damage and sudden death; however, a comprehensive strategy to mitigate this inflammatory response remains elusive. Our study examines the role of NLRP6 in S. suis infection, with a particular focus on its involvement in pathogen regulation. A marked upregulation of NLRP6 was observed in peritoneal macrophages post-infection with S. suis SC19 strain, consequently activating the NLRP6 inflammasome. Furthermore, SC19 infection was found to augment the secretion of pro-inflammatory cytokines IL-1β via NLRP6 activation, while NLRP6 deficiency mitigates the invasion and adhesion of SC19 to macrophages. In vivo models revealed that NLRP6 deletion enhanced survival rates of SC19-infected mice, alongside a reduction in tissue bacterial load and inflammatory cytokine levels. NLRP6 -/- mice were shown to exhibit attenuated inflammatory responses in pulmonary, hepatic, and splenic tissues post-SC19 infection, as evidenced by lower inflammation scores. Flow cytometry analyses further substantiated that NLRP6 is involved in modulating macrophage and neutrophil recruitment during infection. Our findings suggest that NLRP6 negatively regulates host resistance against S. suis infection; its absence results in reduced mortality, bacterial colonization, and a milder inflammatory response. Elucidating the mechanism of NLRP6 in S. suis-induced inflammation provides novel insights and theoretical underpinnings for the prophylaxis and therapeutics of S. suis diseases., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest. No benefits in any form have been or will be received from any commercial party related directly or indirectly to the subject of this manuscript., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Streptococcus suis is an important zoonotic pathogen, causing cytokine storms of Streptococcal toxic shock-like syndrome amongst humans after a wound infection into the bloodstream. To overcome the challenges of fever and leukocyte recruitment, invasive S. suis must deploy multiple stress responses forming a network and utilize proteases to degrade short-lived regulatory and misfolded proteins induced by adverse stresses, thereby adapting and evading host immune responses. In this study, we found that S. suis encodes multiple ATP-dependent proteases, including single-chain FtsH and double-subunit Clp protease complexes ClpAP, ClpBP, ClpCP, and ClpXP, which were activated as the fever of infected mice in vivo. The expression of genes ftsH, clpA/B/C, and clpP, but not clpX, were significantly upregulated in S. suis in response to heat stress, while were not changed notably under the treatments with several other stresses, including oxidative, acidic, and cold stimulation. FtsH and ClpP were required for S. suis survival within host blood under heat stress in vitro and in vivo. Deletion of ftsH or clpP attenuated the tolerance of S. suis to heat, oxidative and acidic stresses, and significantly impaired the bacterial survival within macrophages. Further analysis identified that repressor CtsR directly binds and controls the clpA/B/C and clpP operons and is relieved by heat stress. In summary, the deployments of multiple ATP-dependent proteases form a flexible heat stress response network that appears to allow S. suis to fine-tune the degradation or refolding of the misfolded proteins to maintain cellular homeostasis and optimal survival during infection., (© 2024 Wiley‐VCH GmbH.)

The sit-and-wait hypothesis predicts that bacteria can become more virulent when they survive and transmit outside of their hosts due to circumventing the costs of host mortality. While this hypothesis is largely supported theoretically and through comparative analysis, experimental validation is limited. Here we test this hypothesis in Streptococcus suis , an opportunistic zoonotic pig pathogen, where a pathogenic ecotype proliferated during the change to intensive pig farming that amplifies opportunities for fomite transmission. We show in an in vitro environmental survival experiment that pathogenic ecotypes survive for longer than commensal ecotypes, despite similar rates of decline. The presence of a polysaccharide capsule has no consistent effect on survival. Our findings suggest that extended survival in the food chain may augment the zoonotic capability of S. suis . Moreover, eliminating the long-term environmental survival of bacteria could be a strategy that will both enhance infection control and curtail the evolution of virulence.

Streptococcus suis type 2 (SS2) is a zoonotic pathogen capable of eliciting meningitis, presenting significant challenges to both the swine industry and public health. Suilysin (Sly), one of SS2 most potent virulence determinants, releases a surfeit of inflammatory agents following red blood cell lysis. Notably, while current research on Sly role in SS2-induced meningitis predominantly centers on its interaction with the blood-brain barrier (BBB), the repercussions of Sly hemolytic products on BBB function have largely been sidestepped. In this vein, our study delves into the ramifications of Sly-induced hemolysis on BBB integrity. We discern that Sly hemolytic derivatives exacerbate the permeability of Sly-induced in vitro BBB models. Within these Sly hemolytic products, Interleukin-33 (IL-33) disrupts the expression and distribution of Claudin-5 in brain microvascular endothelial cells, facilitating the release of Interleukin-6 (IL-6) and Interleukin-8 (IL-8), thereby amplifying BBB permeability. Preliminary mechanistic insights suggest that IL-33-driven expression of IL-6 and IL-8 is orchestrated by the p38-mitogen-activated protein kinase signaling, whereas matrix metalloproteinase 9 mediates IL-33-induced suppression of Claudin-5. To validate these in vitro findings, an SS2-infected mouse model was established, and upon intravenous administration of growth stimulation expressed gene 2 (ST2) antibodies, in vivo results further underscored the pivotal role of the IL-33/ST2 axis during SS2 cerebral invasion. In summation, this study pioneerly illuminates the involvement of Sly hemolytic products in SS2-mediated BBB compromise and spotlights the instrumental role and primary mechanism of IL-33 therein. These insights enrich our comprehension of SS2 meningitis pathogenesis, laying pivotal groundwork for therapeutic advancements against SS2-induced meningitis.IMPORTANCEThe treatment of meningitis caused by Streptococcus suis type 2 (SS2) has always been a clinical challenge. Elucidating the molecular mechanisms by which SS2 breaches the blood-brain barrier (BBB) is crucial for the development of meningitis therapeutics. Suilysin (Sly) is one of the most important virulence factors of SS2, which can quickly lyse red blood cells and release large amounts of damage-associated molecular patterns, such as hemoglobin, IL-33, cyclophilin A, and so on. However, the impact of these hemolytic products on the function of BBB is unknown and ignored. This study is the first to investigate the effect of Sly hemolytic products on BBB function. The data are crucial for the study of the pathogenesis of SS2 meningitis and can provide an important reference for the development of meningitis therapeutics., Competing Interests: The authors declare no conflict of interest.

Streptococcus suis (S. suis) is an important gram-positive pathogen and an emerging zoonotic pathogen that causes meningitis in swine and humans. Although several virulence factors have been characterized in S. suis, the underlying mechanisms of pathogenesis are not fully understood. In this study, we identified Zinc metalloproteinase C (ZmpC) probably as a critical virulence factor widely distributed in S. suis strains. ZmpC was identified as a critical facilitator in the development of bacterial meningitis, as evidenced by the detection of increased expression of TNF-α, IL-8, and matrix metalloprotease 9 (MMP-9). Subcellular localization analysis further revealed that ZmpC was localized to the cell wall surface and gelatin zymography analysis showed that ZmpC could cleave human MMP-9. Mice challenge demonstrated that ZmpC provided protection against S. suis CZ130302 (serotype Chz) and ZY05719 (serotype 2) infection. In conclusion, these results reveal that ZmpC plays an important role in promoting CZ130302 to cause mouse meningitis and may be a potential candidate for a S. suis CZ130302 vaccine., (© 2024. The Author(s).)

The subtilisin-like protease-1 (SspA-1) plays an important role in the pathogenesis of a highly virulent strain of Streptococcus suis 2. However, the mechanism of SspA-1-triggered excessive inflammatory response is still unknown. In this study, we demonstrated that activation of type I IFN signaling is required for SspA-1-induced excessive proinflammatory cytokine production. Further experiments showed that the TLR2 endosomal pathway mediates SspA-1-induced type I IFN signaling and the inflammatory response. Finally, we mapped the major signaling components of the related pathway and found that the TIR adaptor proteins Mal, TRAM, and MyD88 and the downstream activation of IRF1 and IRF7 were involved in this pathway. These results explain the molecular mechanism by which SspA-1 triggers an excessive inflammatory response and reveal a novel effect of type I IFN in S. suis 2 infection, possibly providing further insights into the pathogenesis of this highly virulent S. suis 2 strain., Competing Interests: Potential conflicts of interest. All authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2023. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Streptococcus suis is an important pathogen causing severe disease in pigs and humans, giving rise to economic losses in the pig production industry. Out of 65 S. suis isolates collected from diseased pigs in Switzerland between 2019 and 2022, 57 isolates were thoroughly examined by phenotypic and whole genome sequence (WGS) based characterization. The isolates' genomes were sequenced allowing for a comprehensive analysis of their distribution in terms of serovar, sequence type (ST), clonal complex (CC), and classical virulence markers. Antimicrobial resistance (AMR) genes were screened, and phenotypic susceptibility to eight classes of antimicrobial agents was examined. Serovar 6, devoid of any resistance genes, was found to be most prevalent, followed by serovars 1, 3, 1/2, and 9. Thirty STs were identified, with ST1104 being the most prevalent. Serovar 2 and serovar 1/2 were associated with CC1, potentially containing the most virulent variants. Based on single nucleotide polymorphism (SNP) analyses, fifteen isolates belonged to one of seven putative transmission clusters each consisting of two or three isolates. High phenotypic AMR rates were detected for tetracyclines (80%) and macrolides (35%) and associated with the resistance genes tet(O) and erm(B), respectively. In contrast, susceptibility to β-lactam antibiotics and phenicols was high. Determination of phenotypic AMR profiling, including the minimum inhibitory concentrations (MICs) of the tested antimicrobial agents, sets a baseline for future studies. The study provides valuable insights into the genetic diversity and antimicrobial susceptibility of Swiss S. suis isolates, facilitating the identification of emerging clones relevant to public health concerns., 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 B.V. All rights reserved.)

Streptococcus suis (S. suis) is a significant zoonotic microorganism that causes a severe illness in both pigs and humans and is characterized by severe meningitis and septicemia. Suilysin (SLY), which is secreted by S. suis, plays a crucial role as a virulence factor in the disease. To date, the interaction between SLY and host cells is not fully understood. In this study, we identified the interacting proteins between SLY and human brain microvascular endothelial cells (HBMECs) using the TurboID-mediated proximity labeling method. 251 unique proteins were identified in TurboID-SLY treated group, of which six plasma membrane proteins including ARF6, GRK6, EPB41L5, DSC1, TJP2, and PNN were identified. We found that the proteins capable of interacting with SLY are ARF6 and PNN. Subsequent investigations revealed that ARF6 substantially increased the invasive ability of S. suis in HBMECs. Furthermore, ARF6 promoted SLY-induced the activation of p38 MAPK signaling pathway in HBMECs. Moreover, ARF6 promoted the apoptosis in HBMECs through the activation of p38 MAPK signaling pathway induced by SLY. Finally, we confirmed that ARF6 could increase the virulence of SLY in C57BL/6 mice. These findings offer valuable insights that contribute to a deeper understanding of the pathogenic mechanism of SLY., 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.)

Streptococcus suis is an important zoonotic pathogen causing severe infections in pigs and humans. Serotyping of S. suis strains is crucial for epidemiological surveillance, outbreak investigations, and understanding the pathogenesis of this bacterium. Here, we describe a step-by-step approach that enhances a previously developed pipeline by utilizing a computational script for efficient and accurate typing of S. suis strains. The pipeline is implemented in Perl programming language and leverages the Short Read Sequence Typing for Bacterial Pathogens (SRST2) tool. It integrates various bioinformatics techniques and utilizes multiple databases, including a serotype database, cpsH confirmation database, multi-locus sequence typing (MLST) database, recN species-specific gene database, and virulence gene database. These databases contain comprehensive information on S. suis serotypes, genetic markers, and virulence factors. The script can utilize paired-end or single-end fastq files as input and first confirms the species by sequence read data aligning to the recN gene, ensuring the accurate identification of S. suis strains. The pipeline next performs MLST typing and virulence factor identification using SRST2 while in a parallel processes it performs in silico serotyping of the strains. The pipeline offers a streamlined and semiautomated approach to serotyping S. suis strains, facilitating large-scale studies and reducing the manual effort required for data analysis., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Streptococcus suis is a swine bacterial pathogen that predominantly causes disease in weaned piglets characterized by swelling of joints, arthritis, septicemia, meningitis, and sudden death. Intravenous, intramuscular, intraperitoneal, and intranasal infection models were developed to study the bacterial pathogenicity and efficacy of vaccines and various therapeutics. The selection of the appropriate infection model is a critical step in any study, as it may impact the outcomes of the study. Here we describe a method for infecting weaned piglets with S. suis using intraperitoneal route as a reliable, consistent, and reproducible animal model to evaluate vaccine protection against systemic bacterial infection., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Streptococcus suis serotype 2 (SS2) has been reported to be a highly invasive pathogen in swine and a zoonotic agent for humans. Although many bacterial virulence factors have been identified, our an insightful understanding of SS2 pathogenicity is lacking. The gene nadR, encoding nicotinamide-nucleotide adenylyltransferase, was first reported as a regulator and transporter of the intracellular NAD synthesis pathway in Salmonella typhimurium. In this study, we constructed a mutant strain of nadR (ΔnadR) to test the phenotypic and virulence variations between the deletion mutant and the wild-type strain ZY05719. The phenotypic experimental results showed that ΔnadR obviously inhibited cell growth and exhibited shorter chains than WT. The growth defect of ΔnadR was caused by the loss of the function of nadR for transporting the substrates nicotinamide mononucleotide and nicotinamide riboside in the intracellular NAD synthesis pathway. In the process of interaction with the host, ΔnadR participated in adherence and invasion to the host cells, and it was more easily cleared by RAW264.7 macrophages. More importantly, both zebrafish and BALB/c mice in vivo virulence experimental results showed that ΔnadR dramatically attenuated the virulence of SS2, and the ability of ΔnadR to colonize tissues was notably reduced in comparison with that of WT in the BALB/c mice infection model. To the best of our knowledge, this is the first time to demonstrate that nadR not only plays an important role in bacterial growth, but also in connection with the virulence of SS2 as a global transcriptional regulator., Competing Interests: Declaration of Competing Interest None., (Copyright © 2023. Published by Elsevier B.V.)

Streptococcus suis serotype 2 (SS2) is an important zoonotic pathogen that can infect humans in contact with infected pigs or their byproducts. It can employ different types of genes to defend against oxidative stress and ensure its survival. The thioredoxin (Trx) system is a key antioxidant system that contributes adversity adaptation and pathogenicity. SS2 has been shown to encode putative thioredoxin genes, but the biological roles, coding sequence, and underlying mechanisms remains uncharacterized. Here, we demonstrated that SSU05_0237-ORF, from a clinical SS2 strain, ZJ081101, encodes a protein of 104 amino acids with a canonical CGPC active motif and an identity 70-85% similar to the thioredoxin A (TrxA) in other microorganisms. Recombinant TrxA efficiently catalyzed the thiol-disulfide oxidoreduction of insulin. The deletion of TrxA led to a significantly slow growth and markedly compromised tolerance of the pathogen to temperature stress, as well as impaired adhesion ability to pig intestinal epithelial cells (IPEC-J2). However, it was not involved in H 2 O 2 and paraquat-induced oxidative stress. Compared with the wild-type strain, the ΔTrxA strain was more susceptible to killing by macrophages through increasing NO production. Treatment with TrxA mutant strain also significantly attenuated cytotoxic effects on RAW 264.7 cells by inhibiting inflammatory response and apoptosis. Knockdown of pentraxin 3 in RAW 264.7 cells was more vulnerable to phagocytic activity, and TrxA promoted SS2 survival in phagocytic cells depending on pentraxin 3 activity compared with the wild-type strain. Moreover, a co-inoculation experiment in mice revealed that TrxA mutant strain is far more easily cleared from the body than the wild type strain in the period from 8-24 h, and exhibits significantly attenuated oxidative stress and liver injury. In summary, we reveal the important role of TrxA in the pathogenesis of SS2., (© 2023. The Author(s), under exclusive licence to Microbiological Society of Korea.)

Streptococcus suis is a zoonotic pathogen of swine involved in arthritis, polyserositis, and meningitis. Colonization of piglets by S. suis is very common and occurs early in life. The clinical outcome of infection is influenced by the virulence of the S. suis strains and the immunity of the animals. Here, the role of innate immunity was studied in cesarean-derived colostrum-deprived piglets inoculated intranasally with either virulent S. suis strain 10 (S10) or non-virulent S. suis strain T15. Colonization of the inoculated piglets was confirmed at the end of the study by PCR and immunohistochemistry. Fever (≥40.5 °C) was more prevalent in piglets inoculated with S10 compared to T15 at 4 h after inoculation. During the 3 days of monitoring, no other major clinical signs were detected. Accordingly, only small changes in transcription of genes associated with the antibacterial innate immune response were observed at systemic sites, with S10 inducing an earlier response than T15 in blood. Local inflammatory response to the inoculation, evaluated by transcriptional analysis of selected genes in nasal swabs, was more sustained in piglets inoculated with the virulent S10, as demonstrated by transcription of inflammation-related genes, such as IL1B, IL1A, and IRF7. In contrast, most of the gene expression changes in trachea, lungs, and associated lymph nodes were observed in response to the non-virulent T15 strain. Thus, S. suis colonization in the absence of systemic infection induces an innate immune response in piglets that appears to be related to the virulence potential of the colonizing strain., (© 2021. The Author(s).)

Streptococcus suis has been well-recognized as a zoonotic pathogen worldwide, and the diversity and unpredictable adaptive potential of sporadic human strains represent a great risk to the public health. In this study, S. suis LSM178, isolated from a patient in contact with pigs and raw pork, was assessed as a hyper-virulent strain and interpreted for the virulence based on its genetic information. The strain was more invasive for Caco-2 cells than two other S. suis strains, SC19 and P1/7. Sequence analysis designated LSM178 with serotype 2 and a novel sequence type 1005. Phylogenetic analysis showed that LSM178 clustered with highly virulent strains including all human strains and epidemic strains. Compared with other strains, these S. suis have the most and the same virulent factors and a type I-89 K pathogenicity island. Further, groups of genes were identified to distinguish these highly virulent strains from other generally virulent strains, emphasizing the key roles of genes modeling transcription, cell barrier, replication, recombination and repair on virulence regulation. Additionally, LSM178 contains a novel prophage conducive potentially to pathogenicity., (© 2021. The Author(s).)

The epidemic Streptococcus suis ( S. suis ) strain [Sequence type (ST) 7] was gradually evolving from the non-epidemic ST1 strain and got the ability for high expressing of suilysin (SLY). And the high expression of SLY was required for the epidemic strain to cause NLRP3 hyperactivation, which is essential for the induction of cytokines storm, dysfunction of multiple organs, and a high incidence of mortality, the characters of streptococcal toxic shock-like syndrome (STSLS). However, it remains to be elucidated whether acquiring high SLY expression due to genome evolution was sufficient for the non-epidemic strain to cause STSLS. Here, we found that the overexpression of SLY in ST1 strain (P1/7-SLY) could obviously increase the inflammasome activation, which was dependent on NLRP3 signalling. In contrast, the strain (P1/7-mSLY) overexpressing the mutant SLY (protein without hemolytic activity) could not significantly increase the inflammasome activation. Furthermore, similar to the epidemic strain, P1/7-SLY could cause STSLS in nlrp3 +/+ mice but not in nlrp3 -/- mice. In contrast, P1/7-mSLY could not cause STSLS in both nlrp3 +/+ mice and nlrp3 -/- mice. In summary, we demonstrate that genetic evolution enabling S. suis strain to express high level of SLY may be an essential and sufficient condition for NLRP3 inflammasome hyperactivation, which could further cause cytokines storm and STSLS.

Bordetella (B.) bronchiseptica and Streptococcus (S.) suis are major pathogens in pigs, which are frequently isolated from co-infections in the respiratory tract and contribute to the porcine respiratory disease complex (PRDC). Despite the high impact of co-infections on respiratory diseases of swine (and other hosts), very little is known about pathogen-pathogen-host interactions and the mechanisms of pathogenesis. In the present study, we established a porcine precision-cut lung slice (PCLS) model to analyze the effects of B. bronchiseptica infection on adherence, colonization, and cytotoxic effects of S. suis . We hypothesized that induction of ciliostasis by a clinical isolate of B. bronchiseptica may promote subsequent infection with a virulent S. suis serotype 2 strain. To investigate this theory, we monitored the ciliary activity by light microscopy, measured the release of lactate dehydrogenase, and calculated the number of PCLS-associated bacteria. To study the role of the pore-forming toxin suilysin (SLY) in S. suis -induced cytotoxicity, we included a SLY-negative isogenic mutant and the complemented mutant strain. Furthermore, we analyzed infected PCLS by histopathology, immunofluorescence microscopy, and field emission scanning electron microscopy. Our results showed that pre-infection with B. bronchiseptica promoted adherence, colonization, and, as a consequence of the increased colonization, the cytotoxic effects of S. suis , probably by reduction of the ciliary activity. Moreover, cytotoxicity induced by S. suis is strictly dependent on the presence of SLY. Though the underlying molecular mechanisms remain to be fully clarified, our results clearly support the hypothesis that B. bronchiseptica paves the way for S. suis infection.

Streptococcus suis ( S. suis ) is an important rising pathogen that causes serious diseases in humans and pigs. Although some putative virulence factors of S. suis have been identified, its pathogenic mechanisms are largely unclear. Here, we identified a putative virulence-associated factor MutT, which is unique to S. suis serotype 2 (SS2) virulent strains. To investigate the biological roles of MutT in the SS2 virulent strain ZY05719, the mutT knockout mutant (Δ mutT ) was generated and used to explore the phenotypic and virulent variations between the parental and Δ mutT strains. We found that the mutT mutation significantly inhibited cell growth ability, shortened the chain length, and displayed a high susceptibility to H 2 O 2 -induced oxidative stress. Moreover, this study revealed that MutT induced the adhesion and invasion of SS2 to host cells. Deletion of mutT increased microbial clearance in host tissues of the infected mice. Sequence alignment results suggested that mutT was encoded in a strain-specific manner, in which the detection was strongly linked to bacterial pathogenicity. In both zebrafish and mice infection models, the virulence of Δ mutT was largely reduced compared with that of ZY05719. Overall, this study provides compelling evidence that MutT is indispensable for the virulence of SS2 and highlights the biological role of MutT in bacteria pathogenesis during infection.

Streptococcus suis is one of the important emerging zoonotic pathogens. Serotype 2 is most prevalent in patients worldwide. In the present study, we first isolated one S. suis serotype 7 strain GX69 from the blood culture of a patient with septicemia complicated with pneumonia in China. In order to deepen the understanding of S. suis serotype 7 population characteristics, we investigated the phylogenetic structure, genomic features, and virulence of S. suis serotype 7 population, including 35 strains and 79 genomes. Significant diversities were revealed in S. suis serotype 7 population, which were clustered into 22 sequence types (STs), five minimum core genome (MCG) groups, and six lineages. Lineages 1, 3a, and 6 were mainly constituted by genomes from Asia. Genomes of Lineages 2, 3b, and 5a were mainly from Northern America. Most of genomes from Europe (41/48) were clustered into Lineage 5b. In addition to strain GX69, 13 of 21 S. suis serotype 7 representative strains were classified as virulent strains using the C57BL/6 mouse model. Virulence-associated genes preferentially present in highly pathogenic S. suis serotype 2 strains were not suitable as virulence indicators for S. suis serotype 7 strains. Integrative mobilizable elements were widespread and may play a critical role in disseminating antibiotic resistance genes of S. suis serotype 7 strains. Our study confirmed S. suis serotype 7 is a non-negligible pathotype and deepened the understanding of the population structure of S. suis serotype 7, which provided valuable information for the improved surveillance of this serotype.

Mutation rates vary both within and between bacterial species, and understanding what drives this variation is essential for understanding the evolutionary dynamics of bacterial populations. In this study, we investigate two factors that are predicted to influence the mutation rate: ecology and genome size. We conducted mutation accumulation experiments on eight strains of the emerging zoonotic pathogen Streptococcus suis. Natural variation within this species allows us to compare tonsil carriage and invasive disease isolates, from both more and less pathogenic populations, with a wide range of genome sizes. We find that invasive disease isolates have repeatedly evolved mutation rates that are higher than those of closely related carriage isolates, regardless of variation in genome size. Independent of this variation in overall rate, we also observe a stronger bias towards G/C to A/T mutations in isolates from more pathogenic populations, whose genomes tend to be smaller and more AT-rich. Our results suggest that ecology is a stronger correlate of mutation rate than genome size over these timescales, and that transitions to invasive disease are consistently accompanied by rapid increases in mutation rate. These results shed light on the impact that ecology can have on the adaptive potential of bacterial pathogens., Competing Interests: The authors have declared that no competing interests exist.

Streptococcus suis serotype 2 (S. suis 2) is an important zoonotic pathogen that presents a significant threat both to pigs and to workers in the pork industry. The initial steps of S. suis 2 pathogenesis are unclear. In this study, we found that the type II histidine triad protein HtpsC from the highly virulent Chinese isolate 05ZYH33 is structurally similar to internalin A (InlA) from Listeria monocytogenes, which plays an important role in mediating listerial invasion of epithelial cells. To determine if HtpsC and InlA function similarly, an isogenic htpsC mutant (ΔhtpsC) was generated in S. suis by homologous recombination. The htpsC deletion strain exhibited a diminished ability to adhere to and invade epithelial cells from different sources. Double immunofluorescence microscopy also revealed reduced survival of the ΔhtpsC mutant after co-cultivation with epithelium. Adhesion to epithelium and invasion by the wild type strain was inhibited by a monoclonal antibody against E-cadherin. In contrast, the htpsC-deficient mutant was unaffected by the same treatment, suggesting that E-cadherin is the host-cell receptor that interacts with HtpsC and facilitates bacterial internalization. Based on these results, we propose that HtpsC is involved in the process by which S. suis 2 penetrates host epithelial cells, and that this protein is an important virulence factor associated with cell adhesion and invasion., (© 2021. The Microbiological Society of Korea.)

Streptococcus suis is an emerging zoonotic pathogen that causes severe swine and human infections. Metals are essential nutrients for life; however, excess metals are toxic to bacteria. Therefore, maintenance of intracellular metal homeostasis is important for bacterial survival. Here, we characterize a DtxR family metalloregulator, TroR, in S. suis. TroR is located upstream of the troABCD operon, whose expression was found to be significantly downregulated in response to excess manganese (Mn). Deletion of troR resulted in reduced growth when S. suis was cultured in metal-replete medium supplemented with elevated concentrations of zinc (Zn), copper (Cu), or cobalt (Co). Mn supplementation could alleviate the growth defects of the Δ troR mutant under Zn and Co excess conditions; however, it impaired the growth of the wild-type (WT) and complemented (CΔ troR ) strains under Cu excess conditions. The growth of Δ troR was also inhibited in metal-depleted medium supplemented with elevated concentrations of Mn. Moreover, the Δ troR mutant accumulated increased levels of intracellular Mn and Co, rather than Zn and Cu. Deletion of troR in S. suis led to significant upregulation of the troABCD operon. Furthermore, troA expression in the WT strain was induced by ferrous iron [Fe(II)] and Co and repressed by Mn and Cu; the repression of troA was mediated by TroR. Finally, TroR is required for S. suis virulence in an intranasal mouse model. Together, these data suggest that TroR is a negative regulator of the TroABCD system and contributes to resistance to metal toxicity and virulence in S. suis. IMPORTANCE Metals are essential nutrients for life; however, the accumulation of excess metals in cells can be toxic to bacteria. In the present study, we identified a metalloregulator, TroR, in Streptococcus suis, which is an emerging zoonotic pathogen. In contrast to the observations in other species that TroR homologs usually contribute to the maintenance of homeostasis of one or two metals, we demonstrated that TroR is required for resistance to the toxicity conferred by multiple metals in S. suis. We also found that deletion of troR resulted in significant upregulation of the troABCD operon, which has been demonstrated to be involved in manganese acquisition in S. suis. Moreover, we demonstrated that TroR is required for the virulence of S. suis in an intranasal mouse model. Collectively, these results suggest that TroR is a negative regulator of the TroABCD system and contributes to resistance to metal toxicity and virulence in S. suis.

Streptococcus suis serotype 2 (SS2) is an important zoonotic pathogen that poses a serious threat to human health and the swine industry. The survival and travel in the bloodstream are the important causes for SS2, contributing to bacteremia, septicemia even septic shock. However, the related mechanism remains largely unknown. Preliminary experiment demonstrated that SS2 could largely attach to the surface of neutrophils, implying that this phenomenon maybe contributed to the travel of SS2 in bloodstream and then influenced its pathogenicity. To confirm this hypothesis, using a previously established screening method that combines affinity chromatography (based on liquid chromatography-tandem mass spectrometry) with shotgun proteomics, three candidate proteins (HP0487, HP1765, and HP1111) were identified from SS2 that could interact with neutrophils. Next, by constructing the deletion mutations, we demonstrated that HP0487 of three proteins could significantly influence the adhesion of SS2 to neutrophils. Furthermore, HP0487 was shown to contribute to the anti-phagocytosis of SS2 to neutrophils and RAW264.7 cells. More importantly, the deletion of HP0487 significantly reduced lethality and bacterial loads in vivo of SS2. Thus, our findings demonstrate that HP0487 contributes to SS2 virulence by mediating the adhesion and anti-phagocytosis of SS2 to neutrophils, promoting a better understanding about the pathogenesis of SS2., (Copyright © 2021. Published by Elsevier B.V.)

Streptococcus suis serotype (cps) 1 and cps14 have been detected in association with severe diseases such as meningitis and polyarthritis in pigs. Though these two cps are very similar, only cps14 is an important zoonotic agent in Asia and only cps1 is described to be associated with diseases in suckling piglets rather than weaning piglets. The main objective of this study was to assess restriction of survival of cps14 and cps1 in porcine blood by IgG and IgM putatively cross-reacting with these two cps. Furthermore, we differentiate recent European cps1/14 strains by agglutination, cpsK sequencing, MLST and virulence-associated gene profiling. Our data confirmed cps1 of clonal complex 1 as an important pathotype causing polyarthritis in suckling piglets in Europe. The experimental design included also bactericidal assays with blood samples drawn at different ages of piglets naturally infected with different S. suis cps types including cps1 but not cps14. We report survival of a cps1 and a cps14 strain (both of sequence type 1) in blood of suckling piglets with high levels of maternal IgG binding to the bacterial surface. In contrast, killing of cps1 and cps14 was recorded in older piglets due to an increase of IgM as demonstrated by specific cleavage of IgM. Heterologous absorption of antibodies with cps1 or cps14 is sufficient to significantly increase the survival of the other cps. In conclusion, IgM elicited by natural S. suis infection is crucial for killing of S. suis cps1 and cps14 in older weaning piglets and has most likely the potential to cross-react between cps1 and cps14., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The eukaryotic-type serine/threonine kinase of Streptococcus suis serotype 2 (SS2) performs critical roles in bacterial pathogenesis. In this study, isobaric tags for relative and absolute quantification (iTRAQ) MS/MS were used to analyze the protein profiles of wild type strain SS2-1 and its isogenic STK deletion mutant (Δstk). A total of 281 significant differential proteins, including 147 up-regulated and 134 down-regulated proteins, were found in Δstk. Moreover, 69 virulence factors (VFs) among these 281 proteins were predicted by the Virulence Factor Database (VFDB), including 38 downregulated and 31 up-regulated proteins in Δstk, among which 15 down regulated VFs were known VFs of SS2. Among the down-regulated proteins, high temperature requirement A (HtrA), glutamine synthase (GlnA), ferrichrome ABC transporter substrate-binding protein FepB, and Zinc-binding protein AdcA are known to be involved in bacterial survival and/or nutrient and energy acquisition under adverse host conditions. Overall, our results indicate that STK regulates the expression of proteins involved in virulence of SS2 and its adaption to stress environments., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Streptococcus suis (S. suis) is a major bacterial pathogen in the swine industry and an emerging zoonotic agent. S. suis produces an important extracellular component, capsular polysaccharide (CPS), based on which dozens of serotypes have been identified. Through virulence genotyping, we revealed the relatedness between subpopulations of S. suis serotype 2 (SS2), S. suis serotype 3 (SS3) and S. suis serotype 7 (SS7) strains despite their serotype differences. Multilocus sequence typing (MLST) was used to characterize the whole S. suis population and revealed capsule switching between S. suis strains. Importantly, capsule switching occurred in the SS2, SS3 and SS7 strains belonging to CC28 and CC29, which are phylogenetically distinct from the main CC1 SS2 lineage. To further explore capsule switching in S. suis, comparative genomic analyses were performed using available complete S. suis genomes. Phylogenetic analyses suggested that the SS2 strains could be divided into two clades (1 and 2), and those classified into clade 2 colocalized with SS3 and SS7 strains, in accordance with the above virulence genotyping and MLST analyses. Clade 2 SS2 strains presented high genetic similarity to SS3 and SS7 and shared common competence and defensive elements with them but were significantly different from Clade 1 SS2 strains. Notably, although the cps loci shared by Clade 1 and 2 SS2 strains were almost identical, a specific region of the cps locus of strain NSUI002 (Clade 2 SS2) could be found in the SS3 cps locus but not in the Clade 1 SS2 strain. These data indicated that the SS2 strains in CC28 and CC29 might have acquired the cps locus through capsule switching, which could explain the distinct genetic lineages within the SS2 population., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

Streptococcus suis is a zoonotic pathogen that can cause invasive infections in humans and pigs. The S. suis cps31 strains (SS31) were frequently isolated from healthy or diseased pigs and one human infection case caused by SS31 was reported in Thailand in 2015. However, except for a few epidemiologic studies, little information is available for SS31. To characterize SS31, a total of 75 SS31 strains were analyzed, including 52 strains that were isolated from healthy or diseased pigs and 23 strains whose information was accessed from NCBI. The MLST analysis showed that SS31 exhibited high heterogeneity. The phylogenetic analysis and minimum core-genome (MCG) classification revealed that 75 strains were clustered into 3 lineages. Strains from NCBI mainly at Lineage 2 belong to MCG7-3, and most of strains from China at Lineage 3 belong to MCG7-2. This finding indicated that their evolutionary path was different. All SS31 strains were resistant to more than three classes of antimicrobial agents, and major antimicrobial resistance genes for strains from Lineage 3 were carried by prophages. This observation is different from the previous observation that integrative conjugative elements and integrative and mobilizable elements are major vehicles of antimicrobial resistance genes for S. suis. In addition to strains isolated from diseased pigs, seven of 47 strains isolated from clinically healthy pigs were also pathogenic in a zebrafish infection model. These findings reveal unique characteristics of SS31 and contribute to establishing public health surveillance for SS31 and clarifying the diversity of S. suis., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The capsule (cap) of Streptococcus suis is an anti-phagocytic element and is one of the major virulence factors. However, we have found cap-positive and cap-negative isolates in porcine endocarditis. Here, we compared genome sequences of multiple cap-negative isolates with those of a cap-positive isolate from a single endocarditis. Cap-positive and cap-negative isolates from the same pig were phylogenetically closest compared with those from other pigs. Some of cap-negative isolates from the same pig showed different mutations in capsular polysaccharide synthesis (cps) genes, suggesting that these isolates arisen in pigs after infection. Different mutations in whole-genomes were also found among isolates with identical mutations in cps genes, indicating that mutations in cps genes and the whole-genome occurred independently. Since cap-negative isolates are rarely found in lesions of other diseases, these results suggest that endocarditis lesions may simply favored cap-negative mutants to survive the niches, leading to their persistence in the lesions., (© The Author(s) 2021. Published by Oxford University Press on behalf of FEMS. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Streptococcus suis serotype 2 (SS2) is an important zoonotic pathogen that causes serious economic losses in the pig industry. Phosphorylation is an important mechanism of protein modification. Recent studies have reported that the serine/threonine kinase (STK) gene contributes to the growth and virulence of SS2. However, the mechanism underlying the regulatory functions of STK in SS2 has not been thoroughly elucidated to date. In this study, phosphoproteomic analysis was performed to determine substrates of the STK protein. Twenty-two proteins with different cell functions were identified as potential substrates of STK. Phosphoglucosamine mutase (GlmM) was selected for further investigation among them. In vitro phosphorylation assay and immunoprecipitation assay indicated that GlmM was phosphorylated by STK at the Ser-101 site and the phosphorylation level of GlmM can be affected. We observed that compared to the wild-type strain ZY05719, the glmM-deficient strain (ΔglmM) and the glmM S101A point mutation strain (CΔglmM S101A) showed aberrant cell morphology and attenuated virulence, including enlarged cell volume, absent capsule, decreased resistance, lower survival caused by unusual peptidoglycan synthesis, and significantly attenuated pathogenicity in a mouse infection model. Additionally, compared to ZY05719 and CΔglmM, GlmM enzyme acivities and peptidoglycan concentrations of the stk-deficient strain (Δstk), CΔglmM S101A decreased significantly. These experiments revealed that STK phosphorylates GlmM at the Ser-101 site to impact GlmM enzyme activity and control cell wall peptidoglycan synthesis to affect SS2 pathogenicity., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The capsular polysaccharide (CPS) of Streptococcus suis defines various serotypes based on its composition and structure. Though serotype switching has been suggested to occur between S. suis strains, its impact on pathogenicity and virulence remains unknown. Herein, we experimentally generated S. suis serotype-switched mutants from a serotype 2 strain that express the serotype 3, 4, 7, 8, 9, or 14 CPS. The effects of serotype switching were then investigated with regards to classical properties conferred by presence of the serotype 2 CPS, including adhesion to/invasion of epithelial cells, resistance to phagocytosis by macrophages, killing by whole blood, dendritic cell-derived pro-inflammatory mediator production and virulence using mouse and porcine infection models. Results demonstrated that these properties on host cell interactions were differentially modulated depending on the switched serotypes, although some different mutations other than loci of CPS-related genes were found in each the serotype-switched mutant. Among the serotype-switched mutants, the mutant expressing the serotype 8 CPS was hyper-virulent, whereas mutants expressing the serotype 3 or 4 CPSs had reduced virulence. By contrast, switching to serotype 7, 9, or 14 CPSs had little to no effect. These findings suggest that serotype switching can drastically alter S. suis virulence and host cell interactions.

Streptococcus suis serotype 2 (SS2) is an important zoonotic pathogen that causes meningitis. The ubiquitously expressed 40S ribosome protein SA (RPSA) is a multifunctional protein involved in the pathogenesis of multiple pathogens, especially those causing meningitis. However, the role of RPSA in SS2-induced meningitis is not clear. In this study, immunofluorescence staining revealed that SS2 infection promoted the intracellular transfer of RPSA to the surface of human cerebral microvascular endothelial cells (HCMECs). Moreover, SS2 infection promoted the accumulation of caveolin 1 (CAV1) and the formation of membrane bulges where RPSA enveloped CAV1 on the cell surface. SS2 infection also caused dynamic changes in the localization of RPSA and CAV1 on the cell surface which could be eliminated by disruption of caveolae/rafts by addition of methyl-β-cyclodextrin (MβCD). Co-immunoprecipitation analysis demonstrated that α-enolase (ENO), a key virulence factor of SS2, interacted with RPSA, and promoted the interaction between RPSA and CAV1. Immunofluorescence staining, western blotting and flow cytometry analyses showed that damaged caveolae/rafts significantly enhanced ENO adhesion to HCMECs, promoted the "destruction" of RPSA by ENO, and enhanced the toxic effect of ENO on HCMECs. Importantly, these effects could be relieved upon the addition of cholesterol. We conclude that caveolae/rafts weaken the toxic effect of SS2 ENO on RPSA-mediated events in HCMECs. Our study has led to better understanding of the roles of RPSA and caveolae/rafts upon SS2 infection, and a new pathological role for RPSA in infection., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Streptococcus suis (S. suis) is a major zoonotic pathogen and is also responsible for variety of diseases in swine. LytR-CpsA-Psr (LCP) family proteins affect the biofilm formation and virulence of some Gram-positive bacteria, but we know nothing about their roles in S. suis. In this study, we constructed the LytR mutant and its revertant strains by natural transformation and verified them by PCR and western blot. We explored the effects of LytR on the cell morphology of S. suis. Transmission electron microscopic analysis showed that the mutant strain displayed aberrant septum placement with no obvious differences in capsular thickness. Crystal violet staining and laser-scanning confocal microscopy both revealed that LytR contributes to the biofilm formation of S. suis. The LytR mutant strain had reduced survival in whole human blood and was more sensitive to killing by polymorphonuclear leukocytes (PMNs). Furthermore, in a mouse infection model, the LytR mutant strain also exhibited significantly attenuated virulence and was more easily cleared in the blood. These results indicate that the LytR protein is involved in septum placement, biofilm formation and required for full virulence of S. suis during infection., (Copyright © 2021 Elsevier B.V. All rights reserved.)