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

Abstract

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.

Keywords: Streptococcus suis serotype 2; blood bactericidal assay; inflammation; mouse model; phagocytosis; sequence type; virulence.

Figure 1

Timeline summary of the intraperitoneal and intracisternal mouse models of infection used throughout this study. C57BL/6 mice were infected with the different S. suis serotype 2 strains using the intraperitoneal route of infection (A) to evaluate the systemic and subsequent central nervous system infection; or the transcutaneal intracisternal route of infection (B) to directly evaluate the central nervous system infection.

Figure 2

Blood bacterial burden is lower in North American ST28-infected mice but similar in ST1, ST25 or Eurasian ST28-infected mice during the systemic infection. C57BL/6 mice were inoculated by intraperitoneal injection with 5 × 10⁷ CFU and blood bacterial titers evaluated 24 h (A); 36 h (B) and 48 h (C) post-infection (p.i.). Data of individual mice are presented as log₁₀ CFU/mL with the geometric mean. Significance between groups is indicated by different letters (p < 0.001). Only strains for which five or more mice survived at the indicated time point are presented.

Figure 3

Plasma cytokine production is lowest in North American ST28-infected mice, intermediate in Eurasian ST28-infected mice, and highest in ST1- and ST25-infected mice during systemic infection. Plasma cytokine levels 12 h post-intraperitoneal inoculation of mock- (vehicle) or 5 × 10⁷ CFU of S. suis-infected C57BL/6 mice, as determined by Luminex^® for TNF-α (A); IL-6 (B); IL-12p70 (C); and IFN-γ (D). Data of individual mice are presented as pg/mL with the mean. Significance between groups is indicated by different letters (p < 0.05).

Figure 4

Plasma chemokine production is lowest in North American ST28-infected mice, intermediate in Eurasian ST28-infected mice and highest in ST1- and ST25-infected mice during the systemic infection. Plasma chemokine levels 12 h post-intraperitoneal inoculation of mock- (vehicle) or 5 × 10⁷ CFU of S. suis-infected C57BL/6 mice, as determined by Luminex^® for CCL2 (A); CCL3 (B); CCL4 (C); CCL5 (D), and CXCL1 (E). Data of individual mice are presented as pg/mL with the mean. Significance between groups is indicated by different letters (p < 0.05).

Figure 5

Histopathological studies of the brains of C57BL/6 mice infected by intraperitoneal inoculation during central nervous system infection. Presence or absence of histopathological lesions of meningitis as determined in the brains of mock-infected (vehicle) and infected mice. Micrographs of the meninges or ventricular choroid plexus of mock-infected mice (A); NA ST28 strain 1088563- (B); EA ST28 strain MNCM43- (C); NA ST25 strain 89-1591- (D); and EA ST1 strain P1/7- (E) infected mice. Black arrowheads indicate lesions typical of S. suis meningitis. HPS staining, 100× magnification. NA = North America; EA = Eurasia.

Figure 6

Lower virulence North American ST28 strains do not induce meningitis in C57BL/6 mice following intracisternal inoculation. Presence or absence of histopathological lesions of meningitis in the brains of mock-infected (vehicle) and S. suis-infected C57BL/6 mice following intracisternal inoculation. Micrographs of the meninges or ventricular choroid plexus of mock-infected (A); NA ST28 strain 1054471- (B); NA ST28 strain 1088563- (C); and EA ST1 strain P1/7- (D) infected mice. Black arrowheads indicate lesions typical of S. suis meningitis. HPS staining, 100× magnification. NA = North America; EA = Eurasia.

Figure 7

Brain and blood bacterial titers of lower virulence North American ST28 strains are transient, while those of the ST1 strain are persistent, following intracisternal inoculation. C57BL/6 mice were inoculated by intracisternal injection with 2 × 10⁵ CFU and brain and blood bacterial titers evaluated 12 h (A & C) and 24 h (B & D) post-infection (p.i.). Data of individual mice are presented as log₁₀ CFU/g or CFU/mL with the geometric mean. * Indicates a significant difference between the Eurasian ST1 strain P1/7 and both North American ST28 strains (1054471 and 1088563) (p < 0.01).

Figure 8

Plasma (P) and brain (B) homogenate cytokine and chemokines levels of mock-infected (vehicle) or C57BL/6 mice inoculated by intraperitoneal injection with S. suis serotype 2 European ST1 strain P/7, upon presentation of clinical signs of septic shock or meningitis, by ELISA for IL-1β (A); IL-6 (B); CCL2 (C); CCL3 (D); CXCL1 (E); and CXCL10 (F). Data are presented as mean ± SEM pg/mL.

Figure 9

Production of brain cytokines and chemokines following S. suis serotype 2 infection only occurs in the presence of meningitis. Brain cytokine and chemokine levels of mock-infected (vehicle) or S. suis-infected C57BL/6 mice inoculated by intraperitoneal or intracisternal injection as determined by ELISA. Intraperitoneally injected mice were euthanized upon presentation of clinical signs of meningitis or at the end of the study (14 days post-infection) and intracisternally injected mice 24 h post-infection or at the end of the study (72 h post-infection). Brain levels of IL-1β (A); IL-6 (B); CCL2 (C); CCL3 (D); CXCL1 (E); and CXCL10 (F) following infection with EA ST1 strain P1/7, NA ST25 strain 89-1591, NA ST28 strain 1088563, or EA ST28 strain MNCM43. Data are presented as mean ± SEM pg/mL. * Indicates a significant difference with mock-infected mice (p < 0.05). EA = Eurasian; NA = North American.