Multi-omics analysis reveals genes and metabolites involved in Streptococcus suis biofilm formation.

BACKGROUND: Streptococcus suis is an important zoonotic pathogen. Biofilm formation largely explains the difficulty in preventing and controlling S. suis. However, little is known about the molecular mechanism of S. suis biofilm formation. RESULTS: In this study, transcriptomic and metabolomic analyses of S. suis in biofilm and planktonic states were performed to identify key genes and metabolites involved in biofilm formation. A total of 789 differential genes and 365 differential metabolites were identified. By integrating transcriptomics and metabolomics, five main metabolic pathways were identified, including amino acid pathway, nucleotide metabolism pathway, carbon metabolism pathway, vitamin and cofactor metabolism pathway, and aminoacyl-tRNA biosynthesis metabolic pathway. CONCLUSIONS: These results provide new insights for exploring the molecular mechanism of S. suis biofilm formation.

The suppressive role of NLRP6 in host defense against Streptococcus suis infection.

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-1ß 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.

Whole genome sequencing of Streptococcus suis revealed potential drug resistance and zoonotic transmission in companion cat.

Streptococcus suis is a bacterium of clinical importance in diverse animal hosts including companion animals and humans. Companion animals are closely associated in the living environment of humans and are potential reservoirs for zoonotic pathogens. Given the zoonotic potential of S. suis, it is crucial to determine whether this bacterium is present among the companion animal population. This study aimed to detect Streptococcus suis in companion animals namely cats and dogs of the central west coast of Peninsular Malaysia and further characterize the positive isolates via molecular and genomic approach. The detection of S. suis was done via bacterial isolation and polymerase chain reaction assay of gdh and recN gene from oral swabs. Characterization was done by multiplex PCR serotyping, as well as muti-locus sequence typing, AMR gene prediction, MGE identification and phylogenomic analysis on whole genome sequence acquired from Illumina and Oxford Nanopore sequencing. Among the 115 samples, PCR assay detected 2/59 of the cats were positive for S. suis serotype 8 while all screened dog samples were negative. This study further described the first complete whole genome of S. suis strain SS/UPM/MY/F001 isolated from the oral cavity of a companion cat. Genomic analysis revealed a novel strain of S. suis having a unique MLST profile and antimicrobial resistance genes of mefA, msrD, patA, patB and vanY. Mobile genetic elements were described, and pathogenic determinants matched to human and swine strains were identified. Phylogenetic tree analysis on the core genome alignment revealed strain SS/UPM/MY/F001 was distinct from other S. suis strains. This study provided insight into the detection and genomic features of the S. suis isolate of a companion cat and highlighted its potential for antimicrobial resistance and pathogenicity.

In Silico Typing and Identification Confirmation of Isolates.

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.

Detection and Quantification of Conjugative Transfer of Mobile Genetic Elements Carrying Antibiotic Resistance Genes.

Multidrug resistance, due to acquired antimicrobial resistance genes, is increasingly reported in the zoonotic pathogen Streptococcus suis. Most of these resistance genes are carried by chromosomal Mobile Genetic Elements (MGEs), in particular, Integrative and Conjugative Elements (ICEs) and Integrative and Mobilizable Elements (IMEs). ICEs and IMEs frequently form tandems or nested composite elements, which make their identification difficult. To evaluate their mobility, it is necessary to (i) select the suitable donor-recipient pairs for mating assays, (ii) do PCR excision tests to confirm that the genetic element is able to excise from the chromosome as a circular intermediate, and (iii) evaluate the transfer of the genetic element by conjugation by doing mating assays. In addition to a dissemination of resistance genes between S. suis strains, MGEs can lead to a spreading of resistance genes in the environment and toward pathogenic bacteria. This propagation had to be considered in a One Health perspective.

Evaluation of the protective efficacy of three novel identified membrane associated proteins of Streptococcus suis serotype 2.

Streptococcus suis is one of the major pathogens of pigs circulating worldwide, and the development of vaccines will help to effectively control streptococcosis in swine. In this study, we evaluated the potential of three membrane associated proteins, histidine kinase (HK), glycosyltransferase family 2 (Gtf-2) and phosphate binding protein (PsbP) of S. suis as subunit vaccines. Bioinformatics analysis shows that protein ABC is highly conserved in S. suis. To verify the protective effects of these proteins in animal models, recombinant protein HK, Gtf-2 and PsbP were used to immunize BALB/c mice separately. The results showed that these proteins immunization in mice can effectively induce strong humoral immune responses, protect mice from cytokine storms caused by S. suis infection, and have a significant protective effect against lethal doses of S. suis infection. Furthermore, antibodies with opsonic activity exist in the recombinant proteins antiserum to assist phagocytic cells in killing S. suis. Overall, these results indicated that these recombinant proteins all elicit good immune protective effect against S. suis infection and can be represent promising candidate antigens for subunit vaccines against S. suis.

Streptococcus suis serotype 4: a population with the potential pathogenicity in humans and pigs.

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.

Metagenomic next-generation sequencing confirmed a case of sporadic human infection with Streptococcus suis in an urban area.

INTRODUCTION: Streptococcus suis (S. suis) disease is a zoonotic infection caused by invasive S. suis and can lead to meningitis, septic shock, arthritis, and endocarditis. Early treatment is the key to reducing mortality. However, clinical manifestations of most cases are atypical, severely limiting rapid diagnosis and treatment. CASE REPORT: Here, we report a 74-year-old female patient diagnosed with S. suis infection. The main symptoms were hearing loss, lumbago, and scattered ecchymosis of the lower extremities and trunk. Blood non-specific infection indexes were significantly increased and platelets were significantly decreased; however, no pathogens were obtained from routine blood culture. Finally, the S. suis infection was confirmed by metagenomic next-generation sequencing (mNGS) of blood and cerebrospinal fluid. After antibiotic treatment, the limb and trunk scattered ecchymosis and lumbago symptoms were significantly relieved, but the hearing did not recover. CONCLUSIONS: Human infection with S. suis is rare in central cities, and it is easy to misdiagnose, especially in cases with atypical early symptoms. mNGS technology, combined with clinical observation, is helpful to clarify the direction of diagnosis and treatment, which is conducive to patient recovery.

BacScan: a novel genome-wide strategy for uncovering broadly immunogenic proteins in bacteria.

In response to the global threat posed by bacterial pathogens, which are the second leading cause of death worldwide, vaccine development is challenged by the diversity of bacterial serotypes and the lack of immunoprotection across serotypes. To address this, we introduce BacScan, a novel genome-wide technology for the rapid discovery of conserved highly immunogenic proteins (HIPs) across serotypes. Using bacterial-specific serum, BacScan combines phage display, immunoprecipitation, and next-generation sequencing to comprehensively identify all the HIPs in a single assay, thereby paving the way for the development of universally protective vaccines. Our validation of this technique with Streptococcus suis, a major pathogenic threat, led to the identification of 19 HIPs, eight of which conferred 20-100% protection against S. suis challenge in animal models. Remarkably, HIP 8455 induced complete immunity, making it an exemplary vaccine target. BacScan's adaptability to any bacterial pathogen positions it as a revolutionary tool that can expedite the development of vaccines with broad efficacy, thus playing a critical role in curbing bacterial transmission and slowing the march of antimicrobial resistance.

Molecular profile and epidemiological traits of Streptococcus suis isolated from diseased pigs in western Canada reveal multiple-serotype infection: Implications for disease control.

Objective: Streptococcus suis is a major agent of disease in modern swine operations, linked to increased mortality, treatment costs, and secondary infections. Although it is ubiquitous in swine, only a fraction of pigs develop clinical disease. The goals of this study were to profile isolates obtained from diseased pigs in western Canada and to investigate potential associations with disease severity. Procedure: Isolates of S. suis (n = 128) from 75 diagnostic submission and 63 premises were paired with epidemiological surveys completed by submitting practitioners (n = 22). Whole-genome sequencing was used to type isolates. Results: The most prevalent serotypes identified were 1/2 (7.8%, 10/128), 2 (9.3%, 12/128), 3 (9.3%, 12/128), and 7 (7.8%, 10/128); and sequence types 28 (17%, 23/128) and 839 (14%, 19/128). There was no association between serotype or sequence type and organ source or barn location. Approximately 74% (14/19) of the premises had diseased animals colonized by > 1 S. suis serotype, but only 1 pig was simultaneously infected with multiple serotypes and sequence types. Serotype distribution from diseased pigs in western Canada differed from that of those in other geographic regions. Conclusion: Infection of diseased pigs by multiple serotypes should be considered when disease control strategies are implemented. No association between S. suis type and isolation organ was identified.

Le profil moléculaire et les caractéristiques épidémiologiques de Streptococcus suis isolés de porcs malades dans l'ouest du Canada révèlent une infection à sérotypes multiples : implications pour la maitrise de la maladie. Objectif: Streptococcus suis est un agent pathogène majeur dans les exploitations porcines modernes, lié à une mortalité accrue, aux coûts de traitement et aux infections secondaires. Bien qu'elle soit omniprésente chez le porc, seule une fraction des porcs développe une maladie clinique. Les objectifs de cette étude étaient de dresser le profil des isolats obtenus à partir de porcs malades dans l'ouest du Canada et d'étudier les associations potentielles avec la gravité de la maladie. Procédure: Des isolats de S. suis (n = 128) provenant de 75 soumissions pour diagnostic et de 63 sites ont été associés à des enquêtes épidémiologiques réalisées auprès des praticiens soumettant les échantillons (n = 22). Le séquençage du génome entier a été utilisé pour typer les isolats. Résultats: Les sérotypes les plus répandus identifiés étaient 1/2 (7,8 %, 10/128), 2 (9,3 %, 12/128), 3 (9,3 %, 12/128) et 7 (7,8 %, 10/128); et les types de séquence 28 (17 %, 23/128) et 839 (14 %, 19/128). Il n'y avait aucune association entre le sérotype ou le type de séquence et la source d'organes ou l'emplacement de la ferme. Environ 74 % (14/19) des exploitations abritaient des animaux malades colonisés par > 1 sérotype de S. suis, mais 1 seul porc était infecté simultanément par plusieurs sérotypes et types de séquences. La répartition des sérotypes chez les porcs malades de l'ouest du Canada différait de celle des porcs d'autres régions géographiques. Conclusion: L'infection des porcs malades par plusieurs sérotypes doit être envisagée lors de la mise en oeuvre de stratégies de maitrise de la maladie. Aucune association entre le type de S. suis et l'organe d'isolement n'a été identifiée.(Traduit par Dr Serge Messier).

Investigation of genomic and pathogenicity characteristics of Streptococcus suis ST1 human strains from Guangxi Zhuang Autonomous Region (GX) between 2005 and 2020 in China.

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.

Type II and IV toxin-antitoxin systems coordinately stabilize the integrative and conjugative element of the ICESa2603 family conferring multiple drug resistance in Streptococcus suis.

Integrative and conjugative elements (ICEs) play a vital role in bacterial evolution by carrying essential genes that confer adaptive functions to the host. Despite their importance, the mechanism underlying the stable inheritance of ICEs, which is necessary for the acquisition of new traits in bacteria, remains poorly understood. Here, we identified SezAT, a type II toxin-antitoxin (TA) system, and AbiE, a type IV TA system encoded within the ICESsuHN105, coordinately promote ICE stabilization and mediate multidrug resistance in Streptococcus suis. Deletion of SezAT or AbiE did not affect the strain's antibiotic susceptibility, but their duple deletion increased susceptibility, mainly mediated by the antitoxins SezA and AbiEi. Further studies have revealed that SezA and AbiEi affect the genetic stability of ICESsuHN105 by moderating the excision and extrachromosomal copy number, consequently affecting the antibiotic resistance conferred by ICE. The DNA-binding proteins AbiEi and SezA, which bind palindromic sequences in the promoter, coordinately modulate ICE excision and extracellular copy number by binding to sequences in the origin-of-transfer (oriT) and the attL sites, respectively. Furthermore, AbiEi negatively regulates the transcription of SezAT by binding directly to its promoter, optimizing the coordinate network of SezAT and AbiE in maintaining ICESsuHN105 stability. Importantly, SezAT and AbiE are widespread and conserved in ICEs harbouring diverse drug-resistance genes, and their coordinated effects in promoting ICE stability and mediating drug resistance may be broadly applicable to other ICEs. Altogether, our study uncovers the TA system's role in maintaining the genetic stability of ICE and offers potential targets for overcoming the dissemination and evolution of drug resistance.

Genetic diversity and antimicrobial susceptibility of Streptococcus suis from diseased Swiss pigs collected between 2019 - 2022.

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 ß-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.

Identification of plasminogen-binding sites in Streptococcus suis enolase that contribute to bacterial translocation across the blood-brain barrier.

Streptococcus suis is an emerging zoonotic pathogen that can cause invasive disease commonly associated with meningitis in pigs and humans. To cause meningitis, S. suis must cross the blood-brain barrier (BBB) comprising blood vessels that vascularize the central nervous system (CNS). The BBB is highly selective due to interactions with other cell types in the brain and the composition of the extracellular matrix (ECM). Purified streptococcal surface enolase, an essential enzyme participating in glycolysis, can bind human plasminogen (Plg) and plasmin (Pln). Plg has been proposed to increase bacterial traversal across the BBB via conversion to Pln, a protease which cleaves host proteins in the ECM and monocyte chemoattractant protein 1 (MCP1) to disrupt tight junctions. The essentiality of enolase has made it challenging to unequivocally demonstrate its role in binding Plg/Pln on the bacterial surface and confirm its predicted role in facilitating translocation of the BBB. Here, we report on the CRISPR/Cas9 engineering of S. suis enolase mutants eno261, eno252/253/255, eno252/261, and eno434/435 possessing amino acid substitutions at in silico predicted binding sites for Plg. As expected, amino acid substitutions in the predicted Plg binding sites reduced Plg and Pln binding to S. suis but did not affect bacterial growth in vitro compared to the wild-type strain. The binding of Plg to wild-type S. suis enhanced translocation across the human cerebral microvascular endothelial cell line hCMEC/D3 but not for the eno mutant strains tested. To our knowledge, this is the first study where predicted Plg-binding sites of enolase have been mutated to show altered Plg and Pln binding to the surface of S. suis and attenuation of translocation across an endothelial cell monolayer in vitro.

Comparative analysis of the interactions of different Streptococcus suis strains with monocytes, granulocytes and the complement system in porcine blood.

Streptococcus suis (S. suis) is an important porcine pathogen causing meningitis, arthritis, and septicemia. Serotypes 2 and 14 are the most common zoonotic ones worldwide, whereas serotypes 2, 9, and 7 are very important in pigs in Europe. To cause invasive infections S. suis needs to enter the bloodstream. Consequently, the immune response in blood represents an important line of defense and bacteremia plays a key role in the pathogenesis of invasive S. suis infections. We investigated the working hypothesis that S. suis strains of the same serotype but different clonal complex (CC) might exhibit substantial differences in the interaction with components of the immune system in porcine blood. The experimental design of this study includes comparative analysis of 8 virulent strains belonging to 4 serotypes with strains of the same serotype being genetically not closely related. Significant differences between two strains of the same serotype but different clonal complex were recorded in the flow cytometric analysis of association with different leukocytes for serotype 9 and 14. Our results demonstrate that the serotype 9 strain of CC94 shows significantly increased association with monocytes and survival in porcine blood of conventional piglets as well as a tendency towards decreased composition of C3 in plasma of these piglets in comparison to the serotype 9 strain of CC16. Correlation analysis of C3 deposition on the bacterial surface and survival in respective blood samples of 8-week-old piglets demonstrated a negative correlation indicating that C3 deposition is a crucial step to limit bacterial survival and proliferation of different S. suis pathotypes in the blood of these piglets. In summary, our results indicate that the capsule composition of a S. suis strain is not alone sufficient to determine association with leukocytes, activation of complement, induction of proinflammatory cytokines, oxidative burst, and bacterial survival in porcine blood. In this study, substantial differences in these host-pathogen interactions were observed between strains of the same serotype. Therefore, a more comprehensive characterization of the field isolates, including at least MLST analysis to determine the sequence type/clonal complex, is recommended.

Subdural Empyema from Streptococcus suis Infection, South Korea.

In Jeju Island, South Korea, a patient who consumed raw pig products had subdural empyema, which led to meningitis, sepsis, and status epilepticus. We identified Streptococcus suis from blood and the subdural empyema. This case illustrates the importance of considering dietary habits in similar clinical assessments to prevent misdiagnosis.

Transposon library screening to identify genes with a potential role in Streptococcus suis biofilm formation.

Background: Biofilm formation is considered to be one of reasons for difficulty in the prevention and control of Streptococcus suis. Aims: To explore the potential genes involved in the biofilm formation of S. suis. Methods: Transposon mutagenesis technology was used to screen biofilm-defective strains of S. suis, and the potential genes related to biofilm were identified. Results: A total of 19 genes were identified that were involved in bacterial metabolism, peptidoglycan-binding protein, cell wall synthesis, ABC transporters, and so on. Conclusion: This study constructed 979 transposon mutation libraries of S. suis. A total of 19 gene loci related to the formation of S. suis biofilm were identified, providing a reference for exploring the mechanism of S. suis biofilm formation in the future.

Streptococcus suis is an important pathogen (this is a microorganism that causes, or can cause, disease) that can be transmitted between animals and humans. The ability to form a protective community, called a biofilm, is one of the reasons why we can have difficulty in preventing and treating S. suis infection. The main purpose of this study was to screen potential genes that may determine biofilm formation in S. suis. The results revealed 19 genes that may affect the biofilm formation of S. suis.

Characterization of pig tonsils as niches for the generation of Streptococcus suis diversity.

Streptococcus suis is a gram-positive bacterium that causes meningitis, septicemia, endocarditis, and other disorders in pigs and humans. We obtained 42 and 50 S. suis isolates from lesions of porcine endocarditis and palatine tonsils, respectively, of clinically healthy pigs in Japan; we then determined their sequence types (STs) by multilocus sequence typing (MLST), cps genotypes, serotypes, and presence of classical major virulence-associated marker genes (mrp, epf, and sly). The 42 isolates from endocarditis lesions were assigned to a limited number of STs and clonal complexes (CCs). On the other hand, the 50 isolates from tonsils were diverse in these traits and seemingly in the degree of virulence, suggesting that tonsils can accommodate a variety of S. suis isolates. The goeBURST full algorithm using tonsil isolates obtained in this study and those retrieved from the database showed that major CCs as well as many other clusters were composed of isolates originating from different countries, and some of the STs were very similar to each other despite the difference in country of origin. These findings indicate that S. suis with not only different but also similar mutations in the genome have survived in tonsils independently across different geographical locations. Therefore, unlike the lesions of endocarditis, the tonsils of pigs seemingly accommodate various S. suis lineages. The present study suggests that S. suis acquired its diversity by natural mutations during colonization and persistence in the tonsils of pigs.

Loss of expression of the glutamate dehydrogenase (gdh) of Streptococcus suis serotype 2 compromises growth and pathogenicity.

Streptococcus suis serotype 2 is a zoonotic agent that causes substantial economic losses to the swine industry and threatens human public health. Factors that contribute to its ability to cause disease are not yet fully understood. Glutamate dehydrogenase (GDH) is an enzyme found in living cells and plays vital roles in cellular metabolism. It has also been shown to affect pathogenic potential of certain bacteria. In this study, we constructed a S. suis serotype 2 GDH mutant (Δgdh) by insertional inactivation mediated by a homologous recombination event and confirmed loss of expression of GDH in the mutant by immunoblot and enzyme activity staining assays. Compared with the wild type (WT) strain, Δgdh displayed a different phenotype. It exhibited impaired growth in all conditions evaluated (solid and broth media, increased temperature, varying pH, and salinity) and formed cells of reduced size. Using a swine infection model, pigs inoculated with the WT strain exhibited fever, specific signs of disease, and lesions, and the strain could be re-isolated from the brain, lung, joint fluid, and blood samples collected from the infected pigs. Pigs inoculated with the Δgdh strain did not exhibit any clinical signs of disease nor histologic lesions, and the strain could not be re-isolated from any of the tissues nor body fluid sampled. The Δgdh also showed a decreased level of survival in pig blood. Taken together, these results suggest that the gdh is important in S. suis physiology and its ability to colonize, disseminate, and cause disease.

Molecular Epidemiology of Underreported Emerging Zoonotic Pathogen Streptococcus suis in Europe.

Streptococcus suis, a zoonotic bacterial pathogen circulated through swine, can cause severe infections in humans. Because human S. suis infections are not notifiable in most countries, incidence is underestimated. We aimed to increase insight into the molecular epidemiology of human S. suis infections in Europe. To procure data, we surveyed 7 reference laboratories and performed a systematic review of the scientific literature. We identified 236 cases of human S. suis infection from those sources and an additional 87 by scanning gray literature. We performed whole-genome sequencing to type 46 zoonotic S. suis isolates and combined them with 28 publicly available genomes in a core-genome phylogeny. Clonal complex (CC) 1 isolates accounted for 87% of typed human infections; CC20, CC25, CC87, and CC94 also caused infections. Emergence of diverse zoonotic clades and notable severity of illness in humans support classifying S. suis infection as a notifiable condition.