Guillaume Goyette-Desjardins

Streptococcus suis, an important pig pathogen and emerging zoonotic agent-an update on the worldwide distribution based on serotyping and sequence typing.

Streptococcus suis is an important pathogen causing economic problems in the pig industry. Moreover, it is a zoonotic agent causing severe infections to people in close contact with infected pigs or pork-derived products. Although considered sporadic in the past, human S. suis infections have been reported during the last 45 years, with two large outbreaks recorded in China. In fact, the number of reported human cases has significantly increased in recent years. In this review, we present the worldwide distribution of serotypes and sequence types (STs), as determined by multilocus sequence typing, for pigs (between 2002 and 2013) and humans (between 1968 and 2013). The methods employed for S. suis identification and typing, the current epidemiological knowledge regarding serotypes and STs and the zoonotic potential of S. suis are discussed. Increased awareness of S. suis in both human and veterinary diagnostic laboratories and further establishment of typing methods will contribute to our knowledge of this pathogen, especially in regions where complete and/or recent data is lacking. More research is required to understand differences in virulence that occur among S. suis strains and if these differences can be associated with specific serotypes or STs.

Glycoengineered outer membrane vesicles: A novel platform for bacterial vaccines

The World Health Organization has indicated that we are entering into a post-antibiotic era in which infections that were routinely and successfully treated with antibiotics can now be lethal due to the global dissemination of multidrug resistant strains. Conjugate vaccines are an effective way to create a long-lasting immune response against bacteria. However, these vaccines present many drawbacks such as slow development, high price, and batch-to-batch inconsistencies. Alternate approaches for vaccine development are urgently needed. Here we present a new vaccine consisting of glycoengineered outer membrane vesicles (geOMVs). This platform exploits the fact that the initial steps in the biosynthesis of most bacterial glycans are similar. Therefore, it is possible to easily engineer non-pathogenic Escherichia coli lab strains to produce geOMVs displaying the glycan of the pathogen of interest. In this work we demonstrate the versatility of this platform by showing the efficacy of geOMVs as vaccines against Streptococcus pneumoniae in mice, and against Campylobacter jejuni in chicken. This cost-effective platform could be employed to generate vaccines to prevent infections caused by a wide variety of microbial agents in human and animals.

Group B Streptococcus and Streptococcus suis Capsular Polysaccharides Induce Chemokine Production by Dendritic Cells via Toll-Like Receptor 2- and MyD88-Dependent and -Independent Pathways

ABSTRACT Streptococcus agalactiae (also known as group B Streptococcus [GBS]) and Streptococcus suis are encapsulated streptococci causing severe septicemia and meningitis. Bacterial capsular polysaccharides (CPSs) are poorly immunogenic, but anti-CPS antibodies are essential to the host defense against encapsulated bacteria. The mechanisms underlying anti-CPS antibody responses are not fully elucidated, but the biochemistry of CPSs, particularly the presence of sialic acid, may have an immunosuppressive effect. We investigated the ability of highly purified S. suis and GBS native (sialylated) CPSs to activate dendritic cells (DCs), which are crucial actors in the initiation of humoral immunity. The influence of CPS biochemistry was studied using CPSs extracted from different serotypes within these two streptococcal species, as well as desialylated CPSs. No interleukin-1β (IL-1β), IL-6, IL-12p70, tumor necrosis factor alpha (TNF-α), or IL-10 production was observed in S. suis or GBS CPS-stimulated DCs. Moreover, these CPSs exerted immunosuppressive effects on DC activation, as a diminution of gamma interferon (IFN-γ)-induced B cell-activating factor of the tumor necrosis factor family (BAFF) expression was observed in CPS-pretreated cells. However, S. suis and GBS CPSs induced significant production of CCL3, via partially Toll-like receptor 2 (TLR2)- and myeloid differentiation factor 88 (MyD88)-dependent pathways, and CCL2, via TLR-independent mechanisms. No major influence of CPS biochemistry was observed on the capacity to induce chemokine production by DCs, indicating that DCs respond to these CPSs in a patterned way rather than a structure-dedicated manner.

Sialylation of Streptococcus suis serotype 2 is essential for capsule expression but is not responsible for the main capsular epitope.

The capsular polysaccharide is a critical virulence factor of the swine and zoonotic pathogen Streptococcus suis serotype 2. The capsule of this bacterium is composed of five different sugars, including terminal sialic acid. To evaluate the role of sialic acid in the pathogenesis of the infection, the neuC gene, encoding for an enzyme essential for sialic acid biosynthesis, was inactivated in a highly virulent S. suis serotype 2 strain. Using transmission electron microscopy, it was shown that inactivation of neuC resulted in loss of expression of the whole capsule. Compared to the parent strain, the ΔneuC mutant strain was more phagocytosed by macrophages and was also severely impaired in virulence in a mouse infection model. Both native and desialylated S. suis serotype 2 purified capsular polysaccharides were recognized by a polyclonal anti-whole cell S. suis serotype 2 serum and a monospecific polyclonal anti-capsule serotype 2 serum. In contrast, only the native capsular polysaccharide was recognized by a monoclonal antibody specific for the sialic acid moiety of the serotype 2 capsule. Together, our results infer that sialylation of S. suis serotype 2 may be essential for capsule expression, but that this sugar is not the main epitope of this serotype.

Atypical Streptococcus suis in man, Argentina, 2013.

To the Editor: Streptococcus suis is a major swine pathogen and an emerging zoonotic agent that causes mainly meningitis and septic shock (1,2). Among the 35 described serotypes classified by differences in capsular antigens, serotype 2 is the most frequently isolated from humans worldwide, and serotype 14 cases are also increasing in some countries (1). In Southeast Asia, this pathogen affects not only workers in close contact with pig/pork by-products but also the general population, probably because of the widespread presence of backyard types of pig production, open meat markets, and some special dishes prepared with raw meat or blood (3). We report a case of peritonitis caused by an atypical S. suis serotype 21 strain in a patient in Argentina. A 62-year-old man from Santa Fe Province in Argentina, who had a history of tobacco and alcohol abuse, was hospitalized in 2013 as an emergency patient with symptoms of acute abdominal distress. Ten days before admission, abdominal distention, accompanied by intense upper abdominal pain, developed in the patient. The patient’s family reported that he had been having gastrointestinal bleeding 4 days before admission, and he was suspected of having diabetes. At admission, a physical examination indicated jaundice, hepatosplenomegaly, and ascites. A neurologic examination indicated that the patient was conscious, but disoriented, and that his vital signs were stable. The patient had a temperature of 38.9°C, a pulse rate of 130 beats/min, and blood pressure of 110/70 mm Hg. Other laboratory results were a leukocyte count of 2,900 cells/μL (70% neutrophils), a platelet count of 94,000/μL, a serum hemoglobin concentration of 13.20 g/dL, a glucose concentration of 195 mg/dL, a blood urea nitrogen level of 42 mg/dL, a creatinine level of 0.96 mg/dL; a serum bilirubin level of 3.01 mg/dL, an alanine aminotransferase level of 35 U/L, an aspartate aminotransferase level of 70 U/, a serum albumin level of 2.66 g/dL, and an increase in prothrombin time to 22 s. Spontaneous bacterial peritonitis was suspected. Abdominal paracentesis was performed and produced a turbid milky fluid, with a protein level of 1600 mg/dL; 1,340 cells/µL (90% neutrophils), a lactate dehydrogenase level of 221 U/L, and an amylase level of 34 U/L. Samples of blood and ascitic fluid were inoculated into aerobic and anaerobic blood culture bottles. Gram staining was performed and no organisms were observed. Treatment with intravenous ceftriaxone (2g/day) was started after a diagnosis of spontaneous bacterial peritonitis associated with liver cirrhosis was made. After 48 h of incubation, cultures of blood and ascetic fluid were plated onto sheep blood agar and chocolate agar and incubated at 35°C in an atmosphere of 5% CO2. After 24 h of incubation, cultures showed growth of α-hemolytic streptococci. An API Strep Test (bioMerieux, Marcy l’Etoile, France) identified the isolate as S. pneumoniae (probability 58.7%) or S. suis (probability 20.7%). However, these 2 probability values are unacceptable identification confidence levels. Therefore, the species and serotype were identified by sequence analysis of a 16S rRNA gene and a coagglutination test as described (4,5). The isolate was identified as S. suis serotype 21. The infection was considered resolved when all signs and symptoms of infection disappeared, a polymorphonuclear cell count in ascitic fluid decreased to <250 cells/μL, and ascitic fluid cultures were negative for bacteria. Antimicrobial drug therapy was given for 48 h after resolution of the infection. The patient denied any recent occupational or occasional contact with swine or other animals, and he had no history of eating raw or undercooked pork. A biochemically and antigenically atypical strain was isolated from the patient with peritonitis. A reference strain of serotype 21 and most other strains of this serotype had been isolated from tonsils of healthy pigs (6). However, 16 strains had also been isolated from sick pigs during 2008–2011 in Canada (7). These findings indicate that this serotype is potentially virulent. Most strains, including the strain from the patient reported, are usually not identified as S. suis by rapid multitest identification systems (6). There are only 2 reports of S. suis being isolated from humans in Latin America; these reports were also from Argentina (8,9). Because swine production in Argentina is a smaller industry than in other Latin American countries, the higher rate of S. suis isolation rate is probably the consequence of good surveillance systems and awareness of the pathogen by local diagnostic laboratories. The patient did not have any contact with swine, pork-derived products, or raw/undercooked beef. A patient infected with S. suis might be unaware or have no recollection of exposure to animals. Latent infection, with reactivation many years later, has been reported (10). S. suis might become an opportunistic pathogen in persons who are stressed or immunodeficient. This pathogen has also been increasingly isolated from mammals other than pigs and from the environment. The patient in this study had a history of alcohol consumption, which is a reported risk factor for this infection (3).

Explaining the Serological Characteristics of Streptococcus suis Serotypes 1 and 1/2 from Their Capsular Polysaccharide Structure and Biosynthesis.

The capsular polysaccharide (CPS) is a major virulence factor in many encapsulated pathogens, as it is the case for Streptococcus suis, an important swine pathogen and emerging zoonotic agent. Moreover, the CPS is the antigen at the origin of S. suis classification into serotypes. Hence, analyses of the CPS structure are an essential step to dissect its role in virulence and the serological relations between important serotypes. Here, the CPSs of serotypes 1 and 1/2 were purified and characterized for the first time. Chemical and spectroscopic data gave the following repeating unit sequences: [6)[Neu5Ac(α2–6)GalNAc(β1–4)GlcNAc(β1–3)]Gal(β1–3)Gal(β1–4)Glc(β1–]n (serotype 1) and [4)[Neu5Ac(α2–6)GalNAc(β1–4)GlcNAc(β1–3)]Gal(β1–4)[Gal(α1–3)]Rha(β1–4)Glc(β1–]n (serotype 1/2). The Sambucus nigra lectin, which recognizes the Neu5Ac(α2–6)Gal/GalNAc sequence, showed binding to both CPSs. Compared with previously characterized serotype 14 and 2 CPSs, N-acetylgalactosamine replaces galactose as the sugar bearing the sialic acid residue in the side chain. Serological analyses of the cross-reaction of serotype 1/2 with serotypes 1 and 2 and that between serotypes 1 and 14 suggested that the side chain, and more particularly the terminal sialic acid, constitutes one important epitope for serotypes 1/2 and 2. The side chain is also an important serological determinant for serotype 1, yet sialic acid seems to play a limited role. In contrast, the side chain does not seem to be part of a major epitope for serotype 14. These results contribute to the understanding of the relationship between S. suis serotypes and provide the basis for improving diagnostic tools.

Protection Against Streptococcus suis Serotype 2 Infection Using a Capsular Polysaccharide Glycoconjugate Vaccine

ABSTRACT Streptococcus suis serotype 2 is an encapsulated bacterium and one of the most important bacterial pathogens in the porcine industry. Despite decades of research for an efficient vaccine, none is currently available. Based on the success achieved with other encapsulated pathogens, a glycoconjugate vaccine strategy was selected to elicit opsonizing anti-capsular polysaccharide (anti-CPS) IgG antibodies. In this work, glycoconjugate prototypes were prepared by coupling S. suis type 2 CPS to tetanus toxoid, and the immunological features of the postconjugation preparations were evaluated in vivo. In mice, experiments evaluating three different adjuvants showed that CpG oligodeoxyribonucleotide (ODN) induces very low levels of anti-CPS IgM antibodies, while the emulsifying adjuvants Stimune and TiterMax Gold both induced high levels of IgGs and IgM. Dose-response trials comparing free CPS with the conjugate vaccine showed that free CPS is nonimmunogenic independently of the dose used, while 25 μg of the conjugate preparation was optimal in inducing high levels of anti-CPS IgGs postboost. With an opsonophagocytosis assay using murine whole blood, sera from immunized mice showed functional activity. Finally, the conjugate vaccine showed immunogenicity and induced protection in a swine challenge model. When conjugated and administered with emulsifying adjuvants, S. suis type 2 CPS is able to induce potent IgM and isotype-switched IgGs in mice and pigs, yielding functional activity in vitro and protection against a lethal challenge in vivo, all features of a T cell-dependent response. This study represents a proof of concept for the potential of glycoconjugate vaccines in veterinary medicine applications against invasive bacterial infections.

Murine Whole-Blood Opsonophagocytosis Assay to Evaluate Protection by Antibodies Raised Against Encapsulated Extracellular Bacteria

In vaccine development, especially against pathogenic encapsulated extracellular bacteria, functional assays such as the opsonophagocytosis assay (OPA) are preferred to ELISA titers for evaluating protection against infection. Such assays are normally performed using phagocytic cell lines or purified cell types, which underestimate the complexity of blood bactericidal activity. Here, we describe an OPA using murine whole-blood as effector cells, in a small format (0.2 ml), which requires small quantities of sera (80 μl or less) from immunized individuals. Easy to develop and perform, this OPA can be readily adapted to various pathogens and could be used to evaluate sera from human or animal clinical trials of carbohydrate-based vaccines.

A single amino acid polymorphism in the glycosyltransferase CpsK defines four Streptococcus suis serotypes

The capsular polysaccharide (CPS) is the major virulence factor of the emerging zoonotic pathogen Streptococcus suis. CPS differences are also the basis for serological differentiation of the species into 29 serotypes. Serotypes 2 and 1/2, which possess identical gene content in their cps loci, express CPSs that differ only by substitution of galactose (Gal) by N-acetylgalactosamine (GalNAc) in the CPS side chain. The same sugar substitution differentiates the CPS of serotypes 14 and 1, whose cps loci are also identical in gene content. Here, using mutagenesis, CPS structural analysis, and protein structure modeling, we report that a single amino acid polymorphism in the glycosyltransferase CpsK defines the enzyme substrate predilection for Gal or GalNAc and therefore determines CPS composition, structure, and strain serotype. We also show that the different CPS structures have similar antiphagocytic properties and that serotype switching has limited impact on the virulence of S. suis.

Capsular Sialyltransferase Specificity Mediates Different Phenotypes in Streptococcus suis and Group B Streptococcus

The capsular polysaccharide (CPS) represents a key virulence factor for most encapsulated streptococci. Streptococcus suis and Group B Streptococcus (GBS) are both well-encapsulated pathogens of clinical importance in veterinary and/or human medicine and responsible for invasive systemic diseases. S. suis and GBS are the only Gram-positive bacteria which express a sialylated CPS at their surface. An important difference between these two sialylated CPSs is the linkage between the side-chain terminal galactose and sialic acid, being α-2,6 for S. suis but α-2,3 for GBS. It is still unclear how sialic acid may affect CPS production and, consequently, the pathogenesis of the disease caused by these two bacterial pathogens. Here, we investigated the role of sialic acid and the putative effect of sialic acid linkage modification in CPS synthesis using inter-species allelic exchange mutagenesis. To this aim, a new molecular biogenetic approach to express CPS with modified sialic acid linkage was developed. We showed that sialic acid (and its α-2,6 linkage) is crucial for S. suis CPS synthesis, whereas for GBS, CPS synthesis may occur in presence of an α-2,6 sialyltransferase or in absence of sialic acid moiety. To evaluate the effect of the CPS composition/structure on sialyltransferase activity, two distinct capsular serotypes within each bacterial species were compared (S. suis serotypes 2 and 14 and GBS serotypes III and V). It was demonstrated that the observed differences in sialyltransferase activity and specificity between S. suis and GBS were serotype unrestricted. This is the first time that a study investigates the interspecies exchange of capsular sialyltransferase genes in Gram-positive bacteria. The obtained mutants represent novel tools that could be used to further investigate the immunomodulatory properties of sialylated CPSs. Finally, in spite of common CPS structural characteristics and similarities in the cps loci, sialic acid exerts differential control of CPS expression by S. suis and GBS.