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. 2014 Jul 1;210(1):4-13.
doi: 10.1093/infdis/jiu038. Epub 2014 Jan 16.

Emergence of hypervirulent mutants resistant to early clearance during systemic serotype 1 pneumococcal infection in mice and humans

Anna Syk, Martin Norman, Jenny Fernebro, Marilena Gallotta, Susan Farmand, Andreas Sandgren, Staffan Normark, Birgitta Henriques-Normark

Emergence of hypervirulent mutants resistant to early clearance during systemic serotype 1 pneumococcal infection in mice and humans

Anna Syk et al. J Infect Dis. .

Abstract

Background: Streptococcus pneumoniae serotype 1 has a high likelihood of causing invasive disease. Serotype 1 isolates belonging to CC228 are associated with low mortality, while CC217 isolates exhibit high mortality in patients.

Methods: Clinical pneumococcal isolates and mutants were evaluated in wild-type C57BL/6 mice, macrophage-depleted mice, neutrophil-depleted mice, and SIGN-R1 knockout mice. In vitro models included binding and phagocytosis by THP-1 cells, capsule measurements, hydrogen peroxide production, and viability assays.

Results: During early systemic infection in mice with serotype 1, large-colony variants appeared in blood. Similar large colonies were found in blood specimens from patients with invasive disease. Large morphotypes contained higher numbers of viable bacteria, grew faster, produced no or little hydrogen peroxide, and contained mutations in the spxB gene. spxB mutants were considerably more virulent in wild-type mice, less susceptible to early host clearance than wild-type strains after intravenous infection, but impaired in colonization. spxB mutants were less efficiently phagocytosed by macrophages than wild-type bacteria, which, in contrast to spxB mutants, caused more-severe disease when macrophages or SIGN-R1 were depleted.

Conclusions: Hypervirulent spxB mutants are selected in both mice and patients and are resistant to early macrophage-mediated clearance.

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Figures

Figure 1.
Figure 1.
spxB mutant pneumococci of ST228 evoked in mice show large-colony morphotypes and are more virulent in murine intraperitoneal and intravenous infection models. Survival of C57BL/6 mice after infection with pneumococcal strains of serotype 1, ST228; wild-type BHN32ST228, spxB mutant BHN123ST228, and revertant BHN685ST228 (spxB complemented mutant). A, Colony morphology of Streptococcus pneumoniae serotype 1 clinical isolate wild-type BHN32ST228 (smaller colony) and spontaneous mutant BHN123ST228 spxB allele1 (larger colony). B, Survival time after intraperitoneal challenge. Mice infected with wild-type BHN32ST228 (n = 30) and revertant BHN685ST228 (n = 10) survived longer (40 hours and 39 hours, respectively) than those infected with the spontaneous spxB mutant, BHN123ST228 (n = 20), with a median survival time of 15.5 hours. Differences in survival times were statistically significant between mice infected with strains containing functional spxB, compared with the spxB mutants (BHN32ST228 vs BHN123ST228, P < .0001; BHN32ST228 vs BHN685ST228, P = .2631; BHN123ST228 vs BHN685ST228, P < .0001). C, Survival after intravenous challenge. BHN32ST228 (n = 15) and BHN685ST228 (n = 10) showed a longer survival time than mutant BHN123ST228 (n = 10), with median survival times of 103 hours, 150 hours, and 72 hours respectively (BHN32ST228 vs BHN123ST228, P = .0017; BHN32ST228 vs BHN685ST228, P = .5585; BHN123ST228 vs BHN685ST228, P < .0001). Levels of bacteremia are shown after intravenous challenge for wild-type BHN32ST228 (D) revertant BHN685ST228 (E), and mutant BHN123ST228 (F). Abbreviation: CFU, colony-forming unit.
Figure 2.
Figure 2.
spxB mutant strains of ST217, evoked in patients with sepsis, are more virulent in mice after intranasal, and intravenous infection. A, Survival of C57BL/6 mice after intranasal challenge with wild-type BHN682ST217 (n = 20) and spxB mutant BHN683ST217 (n = 20). A significant difference was found in survival between the 2 strains (P = .0099). Levels of bacteremia are shown after intravenous challenge for BHN682ST217 (B) and for BHN683ST217 (C); dashed lines denote the lower limit of detection. D, A significant differences was found in bacterial load in the nasotracheal lavage fluid from surviving mice after intranasal challenge (P = .0313). E, Survival of mice after intravenous challenge with wild-type BHN682ST217 (n = 20) or spxB mutant BHN683ST217 (n = 20). The survival time of BHN683ST217 was shorter (median survival, 7.75 hours), compared with that for BHN682ST217 (24 hours; P < .0001). Level of bacteremia after intravenous challenge with wild-type BHN682ST217 (F) and mutant BHN683ST217 (G). H, Level of bacteremia after intravenous challenge with a mix of wild-type BHN682ST217 and mutant BHN683ST217 in ratio of 99 to 1. Abbreviation: CFU, colony-forming unit.
Figure 3.
Figure 3.
spxB affects the interaction between Streptococcus pneumoniae serotype 1 isolates and the human-derived macrophage cell line THP-1. Wild-type and mutant strains of serotype 1, ST228 (open bars), and ST217 (filled bars) were analyzed in binding and phagocytosis assays, using THP-1 cells. A, spxB mutations affect the binding of S. pneumoniae serotype 1 strains to macrophages. Binding is calculated by subtracting internalized bacteria from cell-associated bacteria, divided by initial inoculum. B, Spontaneous spxB mutant strains (BHN123ST228 and BHN683ST217), as well as the in vitro–generated spxB knockout mutant strain (BHN684ST228), were less phagocytosed by macrophages, compared with the corresponding wild-type strains and the revertant BHN685ST228. The percentage phagocytosis represents the percentage of inoculated bacteria that were internalized. All data are presented as mean ± standard error of the mean. *P < .05; **P < .01; ***P < .001; ****P < .0001. Abbreviations: CFU, colony-forming unit; NS, not significant.
Figure 4.
Figure 4.
In vivo depletion of macrophages by using clodronate leads to increased resistance to early clearance of wild-type pneumococci. Survival of C57BL/6 mice treated with liposome-encapsulated clodronate or mice sham treated with phosphate-buffered saline (PBS) after intravenous challenge with wild-type BHN682ST217 or spxB mutant BHN683ST217. A, Macrophage-depleted mice were more susceptible to spxB-proficient BHN682ST217, compared with nondepleted mice (BHN682ST217 clodronate vs PBS, P = .0005). No significant differences were found in survival between macrophage-depleted mice challenged with wild-type BHN682ST217 or mutant BHN683ST217. Bacteremia levels in mice depleted of macrophages with clodronate (B) or in controls treated with PBS (C) show that macrophage-depleted mice are more resistant to early clearance, compared with wild-type mice inoculated with wild-type spxB-proficient bacteria. Abbreviation: CFU, colony-forming unit.
Figure 5.
Figure 5.
In vivo depletion of neutrophils by using antibody against the Ly-6G surface antigen does not affect early clearance but shortens survival time. Survival of C57BL/6 mice treated with antibody Ly-6G in phosphate-buffered saline (PBS) or mice sham treated with isotype antibody in PBS after intravenous challenge with wild-type BHN682ST217 or spxB mutant BHN683ST217. A, Neutrophil-depleted mice were more susceptible to spxB proficient BHN682ST217, compared with nondepleted mice (BHN682ST217 Ly-6G vs isotype antibody, P = .0001) No significant differences were found in survival between neutrophil-depleted mice and sham-treated mice challenged with spxB mutant BHN683ST217. Bacteremia levels were the same in neutrophil-depleted and sham-treated mice after intravenous challenge with wild-type BHN682ST217 (B) or spxB mutant BHN683ST217 (C). Abbreviation: CFU, colony-forming unit.
Figure 6.
Figure 6.
The C-type lectin SIGN-R1 affects early clearance of serotype 1 pneumococci of ST217 after intravenous infection. A, Survival of wild-type and SIGN-R1–deficient mice after intravenous challenge with wild-type BHN682ST217 or spxB mutant BHN683ST217. SIGN-R1−/− mice infected with BHN682ST217 (n = 24) were more susceptible than wild-type mice infected with the same strain (n = 16), whereas wild-type mice infected with BHN683ST217 (n = 15) were as susceptible as SIGN-R1−/− mice infected with the same strain (n = 17). Bacterial counts (colony-forming units [CFUs]) in the blood in wild-type and SIGNRI−/− mice inoculated with strain BHN682ST217 (B) or BHN683ST217 (C). A higher bacterial load was found in SIGNR1−/− mice infected with BHN682ST217 (open circles), compared with wild-type mice inoculated with the same strain (open squares) at 4 hours (P = .0001), 6 hours (P = .0001), 12 hours (P = .003), and the time mice were euthanized (P = .013). Bacteremia levels were not significantly different between wild-type and SIGNR1−/− mice infected with BHN683ST217.
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