Gut virome derived bacteriophage endolysin Lys572 can effectively control Streptococcus suis in refrigerated and thawed pork.

Streptococcus suis serotype 2 is a significant zoonotic pathogen that poses a serious threat to the health of humans and various animals. Ribosomal protein S1 (RPSA), acting as a key virulence factor, mediates SS2 adhesion to host cells and facilitates its penetration of the blood-brain barrier. In this study, we expressed the SS2 RPSA recombinant protein and used it to immunize BALB/c mice. Through screening, we obtained a high-affinity hybridoma clone and produced a specific anti-RPSA monoclonal antibody (designated mAb-RPSA-5E2). We found that mAb-RPSA-5E2 exhibited potent antibacterial activity, significantly inhibiting the proliferation of SS2 in vitro. In SS2-challenged mice, treatment with mAb-RPSA-5E2 significantly reduced mortality and alleviated pathological damage in the lung and brain tissues. Furthermore, evaluating the immunoprotective effect of the SS2-RPSA protein as a subunit vaccine revealed that this vaccine provided good protection against SS2 infection. These findings indicate that RPSA is a promising candidate target for developing SS2 subunit vaccines and targeted therapeutic biologics. KEY POINTS: • The SS2 RPSA monoclonal antibody can inhibit the proliferation of SS2 in vitro. • The SS2 RPSA monoclonal antibody can reduce the mortality rates of mice. • The SS2-RPSA subunit vaccine provides significant immunoprotective effects in mice.

The sbp3 gene of Streptococcus suis plays an important role in biofilm formation, adhesion, invasion, and cytotoxicity in the human intestinal epithelial cell line.

Portugal faces challenges in implementing the One Health approach for zoonotic disease. To foster integrated solutions, a "One Health Living Lab" initiative engaged diverse stakeholders, focusing on junior professionals (ie, veterinary students, public health residents, and environmental health technicians). For 2 months, multidisciplinary teams addressed Avian influenza H5N1 in cats, Mycobacterium caprae, Streptococcus suis, Crimean-Congo hemorrhagic fever (CCHF) through research, stakeholder engagement and solution codesign. Key outcomes included establishing a feline HPAI sentinel network, confirming S suis occupational risks, developing a timely CCHF preparedness exercise and identifying potential M caprae underdiagnosis. The living lab successfully fostered collaboration and generated practical, bottom-up initiatives. While highlighting persistent systemic barriers like data sharing, the model demonstrates a promising approach for engaging future professionals and strengthening Portugal's health security against zoonotic threats, ensuring sustained support and integration.

Bordetella bronchiseptica is a gram-negative bacterium contributing to respiratory diseases in many different animal species. In the swine population, it occurs frequently and plays a role in the Porcine Respiratory Disease Complex as well as in the pathogenesis of atrophic rhinitis. The dermonecrotic toxin (DNT) is involved in the destruction of the nasal conchae, a hallmark of atrophic rhinitis, and several studies have shown the effects of DNT on osteoblastic cells. Surprisingly, only little is known about the interactions of DNT and respiratory epithelial cells. Thus, we investigated the influence of DNT on porcine respiratory epithelial cells during mono- and co-infections in vitro. For this, we infected porcine precision-cut lung slices and air-liquid interface cultures with a DNT-positive B. bronchiseptica wild-type strain and its isogenic DNT-deficient mutant strain. For co-infection experiments, a Streptococcus suis serotype 2 wild-type strain was used. We evaluated cytotoxic effects and colonization of both pathogens, as well as the pro-inflammatory cytokine response of the host cells. Remarkably, DNT neither contributed to the cytotoxic effects of B. bronchiseptica nor did it affect bacterial colonization. Regarding the cytokine response, pro-inflammatory cytokines were expressed mainly upon infection with B. bronchiseptica but hardly after infection with S. suis, whereas co-infection with both pathogens had an amplifying effect on cytokine expression after prolonged infection, independently of DNT. Concluding, we found no evidence that DNT contributes to the early stages of infection with B. bronchiseptica and S. suis in in vitro models of the porcine respiratory tract.

Penicillin-nonsusceptible Streptococcus suis infections in humans: clinical characteristics and antimicrobial synergy testing.

Streptococcus suis serotype 9 is an emerging zoonotic pathogen threatening pig production and public health. Here, we combined comparative genomics of 16 strains, including clinical isolate SS2401, to elucidate the role of prophages in shaping genomic plasticity and virulence evolution. The pan-genome was open (α = 0.375), highlighting extensive genetic diversity. Prophages were prevalent (56.25% of strains), significantly correlated with larger genomes, and exhibited two integration modes: direct insertion as genomic islands and integration at recombination hotspots associated with large-scale inversions. Phylogenetic analysis of the terminase large subunit (TerL) and whole-genome sequences revealed multiple independent acquisitions, with three prophages in SS2401 originating from distinct lineages ("one strain, multiple sources"). Recombination analysis detected 1,432 events across the core genome, indicating frequent horizontal gene exchange. The virulence gene sly (suilysin) was carried as a gene cassette within prophage Phi2401a. Notably, we identified integrase-deficient but otherwise intact prophages that may function as "gene prisons," stably fixing virulence traits. These findings demonstrate that prophages act as dual drivers of genomic architecture and as dynamic reservoirs for virulence genes, providing a framework for understanding bacterial adaptation and informing surveillance strategies in the swine industry.IMPORTANCEStreptococcus suis serotype 9 poses a significant threat to pig farming and public health worldwide. This study reveals that prophages are not passive passengers but active architects of genomic plasticity and virulence evolution in this pathogen. We demonstrate that prophages contribute to genome expansion, facilitate large-scale chromosomal rearrangements, and carry key virulence genes such as sly. The discovery of a "one strain, multiple sources" acquisition pattern and the "gene prison" model explains how virulence traits can be stably maintained and disseminated. These findings redefine prophages as dual drivers of bacterial evolution, providing new insights for surveillance and intervention strategies in the swine industry.

Large-Scale Landscape of Porcine Respiratory Disease Complex-Associated Pathogens in Spanish Swine Production.

The first documented instance of acute bacterial meningitis caused by Streptococcus suis in Colombia has been reported. A 69‐year‐old male resident of a rural area with a history of direct contact with pigs presented to the clinic with a persistent fever, severe headaches, and altered consciousness. Cerebrospinal fluid analysis showed neutrophilic pleocytosis, increased cerebrospinal fluid protein levels, and decreased glucose due to consumption. Initial culture and confirmation by mass spectrometry MALDI‐TOF identified S. suis, with an MLSB resistance profile and resistance to tetracyclines, but with sensitivity to beta‐lactams, quinolones, and vancomycin. The patient received empirical treatment with ceftriaxone, vancomycin, and ampicillin, with favorable progress from the first 24 h, completing 10 days of guided therapy, restricted to ceftriaxone given the susceptibility profile on the antibiogram and its bioavailability in the central nervous system without immediate sequelae. This finding represents direct evidence of the presence of S. suis in humans in the country, raising the possibility of underdiagnosis associated with a lack of clinical suspicion and limitations in the microbiological capacity of local laboratories. The absence of classic meningeal signs in the clinical presentation underscores the necessity for a high index of suspicion, particularly in rural areas with exposure to pigs. Furthermore, the importance of considering this emerging zoonosis in the differential diagnosis of bacterial meningitis is reinforced, given its potential to cause permanent neurosensory complications. The case study outlined in this text emphasizes the pressing need to reinforce epidemiological surveillance, expand diagnostic capacity, and enhance health personnel’s awareness of this zoonotic pathogen in the region. These measures are crucial for enhancing early detection and optimizing treatment outcomes.

Glycosyltransferases play a pivotal role in regulating biofilm formation and pathogenicity in Streptococcus suis.

Streptococcus suis is a largely neglected but emerging bacterial zoonotic pathogen of global concern for animal welfare, antibiotic resistance development, and human health. No effective vaccines are now available. Here, we identified and characterized the function and structure of two cell wall polysaccharide variants in pathogenic S. suis strains using genetic deletion and (heterologous) complementation, lectin staining, glycan composition analysis, and specialized NMR spectroscopy. Both glycan variants were anionic polymers that differed in the presence of glucose in the side chain as a result of allelic variation in a glycosyltransferase gene. Deletion of this variable glycosyltransferase revealed an identical glycan “core” and affected S. suis morphology and lysozyme resistance. Immunization of pigs with this core domain elicited antibodies that recognized antigenically diverse pathogenic S. suis strains and induced complement deposition on encapsulated pathogenic S. suis strains. This study provides valuable insights for developing next-generation glycoconjugate vaccines, whereby a single-glycan target could protect against the emerging zoonotic pathogen S. suis.

Streptococcus suis (S. suis) induces host cell death and has the ability to invade the blood-brain barrier (BBB). However, S. suis-induced BBB disruption has not been completely elucidated. In this study, we focused on the regulatory role of lysine acetyltransferase 2A (KAT2A) on BBB, which is a key regulator of cell death. We found that S. suis strain SC19 induces cell death in human cerebral microvascular endothelial cell line D3 (hCMEC/D3) by membrane disruption and LDH release. During SC19 infection, KAT2A protein expression was markedly reduced but its mRNA expression was not affected. Further study demonstrated that SC19-induced KAT2A reduction was though ubiquitin-proteasome-mediated protein degradation pathway. KAT2A inhibition using significantly exacerbated SC19-induced cell permeability disruption by transwell infection model. Consistently, KAT2A knockdown and pharmacological inhibition significantly aggravated SC19-induced downregulation of tight junction proteins including ZO-1 and occludin, whereas KAT2A overexpression partially restored their levels. Similarly, KAT2A inhibition aggravated SC19-induced downregulation of ZO-1 in mice brain and increased mice death rate, indicating the protective role of KAT2A on BBB. Mechanistically, KAT2A was found to regulate necroptosis in hCMEC/D3. KAT2A knockdown aggravated necroptosis and upregulated phosphorylation of RIPK1, whereas RIPK1 inhibitor Nec-1 rescued SC19-induced necroptosis, indicating that KAT2A regulates RIPK1-dependent necroptosis during SC19 infection. Furthermore, quantitative proteomic analysis identified a network of putative KAT2A-mediated downstream targets and pathways that contribute to BBB integrity. Collectively, these findings reveal the underlying mechanism by which SC19 disrupts BBB through inducing KAT2A-mediated necroptosis and provide a potential therapeutic target for the treatment of bacterial meningitis.

Streptococcus suis is an emerging zoonotic pathogen that typically causes bacteremia or meningitis in humans, whereas vertebral osteomyelitis with epidural abscess is exceedingly rare and may be missed. We describe a 65-year-old farmer with fever and severe low back pain after long-term bare-handed handling of raw pig lungs. Pre-treatment blood cultures yielded S. suis identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). After transient improvement on empirical therapy, fever recurred with worsening lumbar pain. Contrast-enhanced magnetic resonance imaging (MRI) demonstrated multilevel thoracolumbar pyogenic spondylitis with an epidural abscess and a sub-ligamentous abscess beneath the posterior longitudinal ligament (PLL) extending from L2 to L5. Computed tomography-guided lumbar biopsy followed by tissue metagenomic next-generation sequencing (mNGS) detected S. suis, providing concordant evidence supporting pathogen involvement at the vertebral focus. The bloodstream isolate (SS-JX2025-01) was serotype 2, sequence type 7 (ST7). It remained susceptible to β-lactams and glycopeptides but was resistant to macrolide-lincosamide and tetracycline classes, consistent with erm(B), tet(O), tet(40), and ant(6)-Ia detected by whole-genome sequencing (WGS). Virulence profiling revealed an epf+/sly+/mrp- pattern with multiple adhesins and immune-evasion factors, whereas canonical 89K pathogenicity island markers were absent. Core-genome phylogeny placed SS-JX2025-01 within the Chinese ST7 lineage associated with previous outbreaks. This biopsy-supported case expands the clinical spectrum of invasive S. suis infection, highlights the value of tissue mNGS as an adjunct for supporting deep-seated foci in zoonotic infections, and underscores the importance of occupational prevention in small-scale farming households.

Respiratory bacterial infections represent a major health challenge in swine production, highlighting the need for novel immunomodulatory strategies that enhance host resistance. In this study, we investigated whether porcine intestinal lactobacilli could modulate the gut-lung axis and improve respiratory innate immunity in a mouse model of Streptococcus pneumoniae infection, as a surrogate of Streptococcus suis pneumonia. Three strains of Ligilactobacillus salivarius (LAFF998, LAFF1071, and LAFF1095) were orally administered to Swiss mice prior to pneumococcal challenge. The resistance to the infection, the lung damage and the respiratory innate immune response were evaluated. Only strain LAFF998 significantly reduced pulmonary bacterial loads, prevented bacteremia, and attenuated lung injury. This protective effect was associated with selective modulation of respiratory immunity, characterized by reduced neutrophilic inflammation, increased lymphocyte recruitment, and enhanced activation of alveolar macrophages expressing MHC-II. LAFF998 markedly increased the production of IFN-β, IFN-γ, IL-6, IL-10, and IL-27 in the respiratory tract, without inducing excessive inflammatory damage. Ex vivo and in vitro analyses confirmed that alveolar macrophages from LAFF998-treated mice exhibited a primed phenotype with heightened cytokine responses to pneumococcal stimulation. In contrast, strains LAFF1071 and LAFF1095 failed to confer protection or significantly modulate respiratory immune responses. These findings demonstrate a strict strain-dependent effect among porcine L. salivarius isolates and identify LAFF998 as a potent immunobiotic capable of enhancing respiratory innate immunity through the gut-lung axis. This work supports further studies of LAFF998 as an immunobiotic strategy for the prevention of respiratory infections in pigs.

Streptococcus suis (S. suis) is a zoonotic pathogen that causes severe economic losses in the swine industry and life-threatening infections in humans. The high serotype variability and genomic diversity of S. suis have hindered the development of cross-protective vaccines. Although recent advances in in silico prediction and database-driven antigen discovery have accelerated the development of protein-based vaccines, discrepancies between predicted immunogenicity and experimentally verified protective efficacy in animal models emphasize the need to integrate computational design with empirical validation. Using an in silico-assisted design strategy, predicted T and B cell epitope-rich domains from five S. suis antigens (HP0197, Fnbp, Sao, ScpB, and SLY) were assembled into a multimeric vaccine construct, designated ATOMSSUISpenta, through optimization for predicted immunogenicity, solubility, and allergenicity. Vaccine immunogenicity and efficacy were evaluated in mice through antigen-specific antibody profiling, cellular immunity analysis, and in vivo assessment of protective and cross-serotype immunity. ATOMSSUISpenta elicited strong antigen-specific humoral immune responses against all five component antigens in a mouse model. The vaccine also induced robust Th1- and Th17-type cellular immune responses, which are critical for effective opsonic and mucosal defense against S. suis infection. In addition, we found that ATOMSSUISpenta conferred significant protection in a S. suis serotype 2 infection model and induced opsonic antibody activities against serotypes 4 and 9. These findings highlight the potential of ATOMSSUISpenta as a subunit vaccine strategy with potential for broader protection against S. suis and demonstrate the effectiveness of epitope-based multimeric design in targeting antigenically diverse Gram-positive pathogens.

A two-component system regulating suilysin production in ST1 serotype 2 Streptococcus suis.

Streptococcus suis is an emerging zoonotic porcine pathogen and a leading cause of adult bacterial meningitis in Southeast Asia, associated with raw pork consumption. Most zoonotic S. suis infections globally are caused by strains from lineage CC1 carrying a serotype 2 capsule. However, in Thailand, ∼40% of the reported zoonotic infections are caused by 2 endemic lineages, CC104 and CC233, which also have a serotype 2 capsule. In this study, we aimed to identify the drivers of the emergence and recent evolution of these two lineages. We sequenced the whole genomes of 141 Thai S. suis zoonotic and porcine strains isolated over a 15-year period and combined them with a curated global dataset of 2761 published S. suis genomes. Using comparative genomics, Bayesian evolutionary models, and multivariate analysis, we investigated the emergence of zoonotic potential and multidrug resistance in CC104 and CC233. We estimated recent emergence dates for both CC104 (1990; 95% posterior: 1987-1992) and CC233 (2002; 95% posterior: 2000-2004). Both lineages acquired a serotype capsule 2 from CC1 through a capsule locus switching event, prior to their emergence. Both lineages have also experienced multiple antimicrobial resistance acquisition events, with some strains carrying 12 determinants encoding resistance against 8 classes of antibiotics. Most importantly, CC104 and CC233 lineages are the first zoonotic lineages to have acquired increased resistance to penicillin and ceftriaxone, which form the standard therapy to treat S. suis infections in humans. Horizontal transfer of multiple genomic regions can cause rapid emergence of novel multidrug-resistant zoonotic S. suis lineages. As S. suis is mainly controlled and treated through the use of antibiotics in both pigs and humans, these findings highlight the urgent need for improved and enhanced surveillance, infection control, and treatments.

WGS analysis and virulence of Streptococcus suis serotype 4 ST1689 isolated from an asymptomatic pig, Thailand.

Multidrug resistance (MDR) is frequently evident in Streptococcus suis, generating distinct antimicrobial resistance (AMR) profiles, which limits the effective antimicrobial drug (AMD) options against S. suis in pigs and humans. Despite its significance, there is a lack of studies and pertinent methodologies that uncover complex interactions among AMDs and associated resistance patterns. This study aimed to identify associations between phenotypic resistance patterns of S. suis isolates from swine production systems in the United States against common AMDs using Bayesian network analysis (BNA). Data from 259 unique S. suis isolates collected from 91 farms were included. Phenotypic susceptibility interpretations (resistance vs susceptible) of minimum inhibitory concentrations (MICs) were evaluated for 13 commonly used AMDs: ceftiofur (CEF), penicillin (PEN), enrofloxacin (ENR), gentamicin (GEN), neomycin (NEO), spectinomycin (SPC), sulfadimethoxine (SUL), tiamulin (TIA), tilmicosin (TIL), clindamycin (CLN), chlortetracycline (CHL), oxytetracycline (OXY), and tetracycline (TET). BNA was conducted using the R package bnlearn to identify joint resistance patterns and estimate conditional dependencies among resistance outcomes. Results revealed a high prevalence of MDR: 248 isolates (95.6%) were resistant to more than one AMD, and 209 isolates (80.7%) were resistant to at least one AMD in three or more classes. The Bayesian network comprised of 11 edges connecting 13 AMD nodes, highlighting statistical dependencies between AMDs resistances. PEN, TIA, and TIL were the most central nodes, with PEN connected to SUL, TIA, GEN, and CEF; TIA to PEN, SPC, TIL, and CLN; and TIL to SUL, TIA, CLN, and OXY. Other associations included CEF-SPC, TET-CLN, CEF-ENR, and OXY-CHL. These relationships implicate systematic dependencies between AMDs and may have resulted from mechanisms like cross-resistance and co-resistance. While these relationships are statistically derived and hypothesis-generating, they underscore the importance of understanding AMR patterns in guiding more effective AMD use. This approach can help prevent overuse, reduce treatment failures, and support AMR mitigation efforts for improved animal and public health outcomes.

Diagnostic and pathological characterization of senecavirus A-associated epidemic transient neonatal losses in swine.