Jean-Philippe Auger

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.

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).

Virulence Studies of Different Sequence Types and Geographical Origins of Streptococcus suis Serotype 2 in a Mouse Model of Infection.

Multilocus sequence typing previously identified three predominant sequence types (STs) of Streptococcus suis serotype 2: ST1 strains predominate in Eurasia while North American (NA) strains are generally ST25 and ST28. However, ST25/ST28 and ST1 strains have also been isolated in Asia and NA, respectively. Using a well-standardized mouse model of infection, the virulence of strains belonging to different STs and different geographical origins was evaluated. Results demonstrated that although a certain tendency may be observed, S. suis serotype 2 virulence is difficult to predict based on ST and geographical origin alone; strains belonging to the same ST presented important differences of virulence and did not always correlate with origin. The only exception appears to be NA ST28 strains, which were generally less virulent in both systemic and central nervous system (CNS) infection models. Persistent and high levels of bacteremia accompanied by elevated CNS inflammation are required to cause meningitis. Although widely used, in vitro tests such as phagocytosis and killing assays require further standardization in order to be used as predictive tests for evaluating virulence of strains. The use of strains other than archetypal strains has increased our knowledge and understanding of the S. suis serotype 2 population dynamics.

Complex Population Structure and Virulence Differences among Serotype 2 Streptococcus suis Strains Belonging to Sequence Type 28.

Streptococcus suis is a major swine pathogen and a zoonotic agent. Serotype 2 strains are the most frequently associated with disease. However, not all serotype 2 lineages are considered virulent. Indeed, sequence type (ST) 28 serotype 2 S. suis strains have been described as a homogeneous group of low virulence. However, ST28 strains are often isolated from diseased swine in some countries, and at least four human ST28 cases have been reported. Here, we used whole-genome sequencing and animal infection models to test the hypothesis that the ST28 lineage comprises strains of different genetic backgrounds and different virulence. We used 50 S. suis ST28 strains isolated in Canada, the United States and Japan from diseased pigs, and one ST28 strain from a human case isolated in Thailand. We report a complex population structure among the 51 ST28 strains. Diversity resulted from variable gene content, recombination events and numerous genome-wide polymorphisms not attributable to recombination. Phylogenetic analysis using core genome single-nucleotide polymorphisms revealed four discrete clades with strong geographic structure, and a fifth clade formed by US, Thai and Japanese strains. When tested in experimental animal models, strains from this latter clade were significantly more virulent than a Canadian ST28 reference strain, and a closely related Canadian strain. Our results highlight the limitations of MLST for both phylogenetic analysis and virulence prediction and raise concerns about the possible emergence of ST28 strains in human clinical cases.

IRF-8 regulates expansion of myeloid-derived suppressor cells and Foxp3+ regulatory T cells and modulates Th2 immune responses to gastrointestinal nematode infection

Interferon regulatory factor-8 (IRF-8) is critical for Th1 cell differentiation and negatively regulates myeloid cell development including myeloid-derived suppressor cells (MDSC). MDSC expand during infection with various pathogens including the gastrointestinal (GI) nematode Heligmosomoides polygyrus bakeri (Hpb). We investigated if IRF-8 contributes to Th2 immunity to Hpb infection. Irf8 expression was down-regulated in MDSC from Hpb-infected C57BL/6 (B6) mice. IRF-8 deficient Irf8-/- and BXH-2 mice had significantly higher adult worm burdens than B6 mice after primary or challenge Hpb infection. During primary infection, MDSC expanded to a significantly greater extent in mesenteric lymph nodes (MLN) and spleens of Irf8-/- and BXH-2 than B6 mice. CD4+GATA3+ T cells numbers were comparable in MLN of infected B6 and IRF-8 deficient mice, but MLN cells from infected IRF-8 deficient mice secreted significantly less parasite-specific IL-4 ex vivo. The numbers of alternatively activated macrophages in MLN and serum levels of Hpb-specific IgG1 and IgE were also significantly less in infected Irf8-/- than B6 mice. The frequencies of antigen-experienced CD4+CD11ahiCD49dhi cells that were CD44hiCD62L- were similar in MLN of infected Irf8-/- and B6 mice, but the proportions of CD4+GATA3+ and CD4+IL-4+ T cells were lower in infected Irf8-/- mice. CD11b+Gr1+ cells from naïve or infected Irf8-/- mice suppressed CD4+ T cell proliferation and parasite-specific IL-4 secretion in vitro albeit less efficiently than B6 mice. Surprisingly, there were significantly more CD4+ T cells in infected Irf8-/- mice, with a higher frequency of CD4+CD25+Foxp3+ T (Tregs) cells and significantly higher numbers of Tregs than B6 mice. In vivo depletion of MDSC and/or Tregs in Irf8-/- mice did not affect adult worm burdens, but Treg depletion resulted in higher egg production and enhanced parasite-specific IL-5, IL-13, and IL-6 secretion ex vivo. Our data thus provide a previously unrecognized role for IRF-8 in Th2 immunity to a GI nematode.

Streptococcus suis serotype 2 strains isolated in Argentina (South America) are different from those recovered in North America and present a higher risk for humans

Introduction: Streptococcus suis serotype 2 is an important swine pathogen and emerging zoonotic agent causing meningitis and septicemia/septic shock. Strains are usually virulent (Eurasia) or of intermediate/low virulence (North America). Very few data regarding human and swine isolates from South America are available. Case presentation: Seventeen new human S. suis cases in Argentina (16 serotype 2 strains and a serotype 5 strain) are reported. Alongside, 14 isolates from pigs are analyzed: 12 from systemic disease, one from lungs and one from tonsils of a healthy animal. All human serotype 2 strains and most swine isolates are sequence type (ST) 1, as determined by multilocus sequence typing and present a mrp+/epf+/sly+ genotype typical of virulent Eurasian ST1 strains. The remaining two strains (recovered from swine lungs and tonsils) are ST28 and possess a mrp+/epf−/sly− genotype typical of low virulence North American strains. Representative human ST1 strains as well as one swine ST28 strain were analyzed by whole-genome sequencing and compared with genomes from GenBank. ST1 strains clustered together with three strains from Vietnam and this cluster is close to another one composed of 11 strains from the United Kingdom. Conclusion: Close contact with pigs/pork products, a good surveillance system, and the presence of potentially virulent Eurasian-like serotype 2 strains in Argentina may be an important factor contributing to the higher number of human cases observed. In fact, Argentina is now fifth among Western countries regarding the number of reported human cases after the Netherlands, France, the UK and Poland.

Streptococcus suis Serotype 2 Capsule In Vivo

Many Streptococcus suis isolates from porcine endocarditis in slaughterhouses have lost their capsule and are considered avirulent. However, we retrieved capsule- and virulence-recovered S. suis after in vivo passages of a nonencapsulated strain in mice, suggesting that nonencapsulated S. suis are still potentially hazardous for persons in the swine industry.

Serotype-specific role of antigen I/II in the initial steps of the pathogenesis of the infection caused by Streptococcus suis

Streptococcus suis is one of the most important post-weaning porcine bacterial pathogens worldwide. The serotypes 2 and 9 are often considered the most virulent and prevalent serotypes involved in swine infections, especially in Europe. However, knowledge of the bacterial factors involved in the first steps of the pathogenesis of the infection remains scarce. In several pathogenic streptococci, expression of multimodal adhesion proteins known as antigen I/II (AgI/II) have been linked with persistence in the upper respiratory tract and the oral cavity, as well as with bacterial dissemination. Herein, we report expression of these immunostimulatory factors by S. suis serotype 2 and 9 strains and that AgI/II-encoding genes are carried by integrative and conjugative elements. Using mutagenesis and different in vitro assays, we demonstrate that the contribution of AgI/II to the virulence of the serotype 2 strain used herein appears to be modest. In contrast, data demonstrate that the serotype 9 AgI/II participates in self-aggregation, induces salivary glycoprotein 340-related aggregation, contributes to biofilm formation and increased strain resistance to low pH, as well as in bacterial adhesion to extracellular matrix proteins and epithelial cells. Moreover, the use of a porcine infection model revealed that AgI/II contributes to colonization of the upper respiratory tract of pigs. Taken together, these findings suggest that surface exposed AgI/II likely play a key role in the first steps of the pathogenesis of the S. suis serotype 9 infection.

The bias of experimental design, including strain background, in the determination of critical Streptococcus suis serotype 2 virulence factors

Streptococcus suis serotype 2 is an important porcine bacterial pathogen and emerging zoonotic agent mainly responsible for sudden death, septic shock, and meningitis. However, serotype 2 strains are genotypically and phenotypically heterogeneous. Though a multitude of virulence factors have been described for S. suis serotype 2, the lack of a clear definition regarding which ones are truly “critical” has created inconsistencies that have only recently been highlighted. Herein, the involvement of two factors previously described as being critical for S. suis serotype 2 virulence, whether the dipeptidyl peptidase IV and autolysin, were evaluated with regards to different ascribed functions using prototype strains belonging to important sequence types. Results demonstrate a lack of reproducibility with previously published data. In fact, the role of the dipeptidyl peptidase IV and autolysin as critical virulence factors could not be confirmed. Though certain in vitro functions may be ascribed to these factors, their roles are not unique for S. suis, probably due to compensation by other factors. As such, variations and discrepancies in experimental design, including in vitro assays, cell lines, and animal models, are an important source of differences between results. Moreover, the use of different sequence types in this study demonstrates that the role attributed to a virulence factor may vary according to the S. suis serotype 2 strain background. Consequently, it is necessary to establish standard experimental designs according to the experiment and purpose in order to facilitate comparison between laboratories. Alongside, studies should include strains of diverse origins in order to prevent erroneous and biased conclusions that could affect future studies.

Limited Interactions between Streptococcus Suis and Haemophilus Parasuis in In Vitro Co-Infection Studies

Streptococcus suis and Haemophilus parasuis are normal inhabitants of the porcine upper respiratory tract but are also among the most frequent causes of disease in weaned piglets worldwide, causing inflammatory diseases such as septicemia, meningitis and pneumonia. Using an in vitro model of infection with tracheal epithelial cells or primary alveolar macrophages (PAMs), it was possible to determine the interaction between S. suis serotype 2 and H. parasuis strains with different level of virulence. Within H. parasuis strains, the low-virulence F9 strain showed higher adhesion levels to respiratory epithelial cells and greater association levels to PAMs than the high-virulence Nagasaki strain. Accordingly, the low-virulence F9 strain induced, in general, higher levels of pro-inflammatory cytokines than the virulent Nagasaki strain from both cell types. In general, S. suis adhesion levels to respiratory epithelial cells were similar to H. parasuis Nagasaki strain. Yet, S. suis strains induced a significantly lower level of pro-inflammatory cytokine expression from epithelial cells and PAMs than those observed with both H. parasuis strains. Finally, this study has shown that, overall and under the conditions used in the present study, S. suis and H. parasuis have limited in vitro interactions between them and use probably different host receptors, regardless to their level of virulence.