Clinical Relevance and Molecular Pathogenesis of the Emerging Serotypes 22F and 33F of Streptococcus pneumoniae in Spain

Abstract

Streptococcus pneumoniae is the main bacterial cause of respiratory infections in children and the elderly worldwide. Serotype replacement is a frequent phenomenon after the introduction of conjugated vaccines, with emerging serotypes 22F and 33F as frequent non-PCV13 serotypes in children and adults in North America and other countries. Characterization of mechanisms involved in evasion of the host immune response by these serotypes is of great importance in public health because they are included in the future conjugated vaccines PCV15 and PCV20. One of the main strategies of S. pneumoniae to persistently colonize and causes infection is biofilm formation. In this study, we have evaluated the influence of capsule polysaccharide in biofilm formation and immune evasion by using clinical isolates from different sources and isogenic strains with capsules from prevalent serotypes. Since the introduction of PCV13 in Spain in the year 2010, isolates of serotypes 22F and 33F are rising among risk populations. The predominant circulating genotypes are ST433^22F and ST717^33F , being CC433 in 22F and CC717 in 33F the main clonal complexes in Spain. The use of clinical isolates of different origin, demonstrated that pediatric isolates of serotypes 22F and 33F formed better biofilms than adult isolates and this was statistically significant. This phenotype was greater in clinical isolates from blood origin compared to those from cerebrospinal fluid, pleural fluid and otitis. Opsonophagocytosis assays showed that serotype 22F and 33F were recognized by the PSGL-1 receptor on leukocytes, although serotype 22F, was more resistant than serotype 33F to phagocytosis killing and more lethal in a mouse sepsis model. Overall, the emergence of additional PCV15 serotypes, especially 22F, could be associated to an enhanced ability to divert the host immune response that markedly increased in a biofilm state. Our findings demonstrate that pediatric isolates of 22F and 33F, that form better biofilm than isolates from adults, could have an advantage to colonize the nasopharynx of children and therefore, be important in carriage and subsequent dissemination to the elderly. The increased ability of serotype 22F to avoid the host immune response, might explain the emergence of this serotype in the last years.

Keywords: PCV-pneumococcal conjugate vaccine; PSGL-1; Streptococcus pneumoniae; biofilms; serotype 22F; serotype 33F.

FIGURE 1

Trends in IPD incidence due to serotypes 22F and 33F producing IPD episodes in the pediatric population during the period 2009–2018 in Spain. (A) Number of IPD cases. (B) Incidence of IPD cases. Green line with dots is the evolution of serotype 22F whereas red line with squares is the evolution of serotype 33F.

FIGURE 2

Trends in IPD incidence due to serotypes 22F and 33F producing IPD episodes in adults during the period 2009–2018 in Spain. (A) Number of IPD cases of serotype 22F. (B) Incidence of IPD cases of serotype 22F. (C) Number of IPD cases of serotype 33F. (D) Incidence of IPD cases of serotype 33F. Black line with triangles is the evolution in adults of all ages. Green line with dots is the evolution in adults 18–64 years. Red line with squares is the evolution in adults ≥65 years.

FIGURE 3

Biofilm formation of pediatric and adult clinical isolates from four different origins. (A) Represents clinical isolates of serotype 22F (B). Represents clinical isolates of serotypes 33F. M11, non-encapsulated strain; P244, 22F isogenic transformant; P017, 33F isogenic transformant; P, pediatric isolates; A, adult isolates; CSF, cerebrospinal fluid; PF, pleural fluid. Black bars represent growth and pattern or pattern-less bars represent biofilm formation. All data were normalized to the A₅₉₅ CV blanks. Error bars represent standard deviations, and asterisks mark results that are statistically significant (two-tailed Student’s t-test: *P < 0.05; **P < 0.01; ***P < 0.001).

FIGURE 4

Analysis of biofilm formation of pediatric and adult clinical isolates. (A) Represents the comparison between adult and pediatric clinical isolates from four different origins: 22F (left) and 33F (right). (B) Represents the comparison between 8 additional adult and pediatric clinical isolates from blood: 22F (left) and 33F (right). M11, non-encapsulated strain; P244, 22F isogenic transformant; P017, 33F isogenic transformant. Black bars represent growth and white or color bars represent biofilm formation. All data were normalized to the A₅₉₅ CV blanks. Error bars represent standard deviations, and asterisks mark results that are statistically significant (two-tailed Student’s t-test: ^∗P < 0.05; ^∗∗P < 0.01; ^∗∗∗P < 0.001).

FIGURE 5

Influence of different CPS in the evasion of opsonophagocytosis. (A) Bacterial survival of control strain M11 expressing different CPS and grown as a planktonic culture. (B) Bacterial survival of control strain M11 expressing different CPS and grown as a biofilm. Error bars represent standard deviations, and asterisks mark results that are statistically significant (two-tailed Student’s t-test: *P < 0.05; **P < 0.01; ***P < 0.001).

FIGURE 6

Evasion of opsonophagocytosis of 22F and 33F clinical isolates. (A) Bacterial survival of clinical strains from pediatric (P) and adult (A) patients and grown as a planktonic culture. (B) Bacterial survival of clinical strains from pediatric (P) and adult (A) patients and grown as a biofilm. Error bars represent standard deviations, and asterisks mark results that are statistically significant (two-tailed Student’s t-test: ***P < 0.001).

FIGURE 7

Phagocytosis mediated by PSGL-1 receptor. Results represent bacterial survival of control strain M11 expressing different CPS and grown as a planktonic culture. Phagocytic cells were incubated with an IgG isotype negative control that does not block PSGL-1 (black bars), whereas cells exposed to the KPL-1 antibody had the PSGL-1 blocked (hatched bars). Error bars represent standard deviations, and asterisks mark results that are statistically significant (two-tailed Student’s t-test: ***P < 0.001).

FIGURE 8

Virulence of isolates of serotypes 22F and 33F in a sepsis model of infection. (A) Colony counts expressed as Log₁₀ CFU/ml from the blood of mice infected with M11 strain expressing serotype 22F (P244) or serotype 33F (P017) or with clinical isolates of serotypes 22F or 33F from pediatric origin (P) or adult origin (A). (B) Survival curves of P244 vs. P017 strains. (C) Survival curves of clinical isolates of serotype 33F from pediatric origin (P) or adult (A). Error bars represent standard deviations, and asterisks mark results that are statistically significant (two-tailed Student’s t-test: *P < 0.05; ***P < 0.001 for bacterial counts; and Log-rank test *P < 0.05; **P < 0.01 for survival curves).