Emergence of Hypervirulent Mutants Resistant to Early Clearance During Systemic Serotype 1 Pneumococcal Infection in Mice and Humans

(See the editorial commentary by Hakansson on pages 1–3.) Streptococcus pneumoniae is a commensal pathogen adapted to its main ecological niche, the nasopharynx of preschool-aged children. Even though cases of healthy colonization vastly outnumber cases of invasive disease, S. pneumoniae is one of the most common causes of community-acquired pneumonia, sepsis, and meningitis worldwide. It is not known whether colonizing pneumococci appear in the bloodstream genetically unaltered or whether pathoadaptive mutations are selected for in the host that may enhance a given strain's capability of causing invasive disease. Pneumococci comprise a large number of clonal types that may express one of at least 93 different capsular serotypes that differ in their odds ratio of causing invasive disease. Serotype 1 pneumococci have been found to exhibit a high invasive disease potential [1, 2] and cause infections among previously healthy individuals [3]. Serotype 1 isolates belong to the top 5 serotypes associated with invasive pneumococcal disease (IPD) in Europe, Asia, Africa, and Oceania [4]. Despite a high attack rate, disease severity has been reported by us and others to be relatively low, with low or no case-fatality rates [1, 3, 5]. However, epidemic outbreaks of pneumococcal meningitis caused by serotype 1 in Burkina Faso and Ghana have been reported to be associated with a high mortality (44%–74%). European serotype 1 isolates belong predominantly to CC228 (including ST227/228/306), whereas the Burkina Faso and Ghana isolates belonged to CC217 [6–8]. Pneumococci are unusual in that they produce high, potentially suicidal levels of hydrogen peroxide by converting pyruvate to acetyl phosphate and hydrogen peroxide via a pyruvate oxidase encoded by the spxB gene [9]. Hydrogen peroxide production causes bacterial death in the absence of exogenously added catalase. Therefore, spxB mutants tend to grow faster and form considerably larger colonies than wild-type bacteria on blood agar plates. It has been demonstrated that hydrogen peroxide–producing wild-type strains provide a competitive advantage over spx-mutant derivatives in animal colonization models [9–14]. Pneumococcal hydrogen peroxide production has also been shown to be important for outcompeting Staphylococcus aureus from the nose in mixed infections [15, 16]. Thus, during normal colonization the advantage of producing hydrogen peroxide outweighs the growth defect associated with its toxicity. In this article, we demonstrate that spxB mutants emerge spontaneously as large colonies after culturing blood specimens from infected patients and from mice with invasive disease caused by serotype 1 pneumococci of CC228 and CC217. These hydrogen peroxide–deficient mutants are considerably more virulent in mice because of reduced early clearance but appear to be less successful in colonization. Also, we show that early resistance to eradication of spxB mutant bacteria in vivo is due to increased resistance to macrophage-mediated clearance.