Antibiotic-tolerant persisters are pervasive among clinical Streptococcus pneumoniae isolates and show strong condition-dependence

Abstract

Streptococcus pneumoniae is an important human pathogen, being one of the most common causes of community-acquired pneumonia and otitis media. Antibiotic resistance in S. pneumoniae is an emerging problem as it depletes our arsenal of effective drugs. In addition, persistence also contributes to the antibiotic crisis in many other pathogens, yet, in S. pneumoniae nothing is known about antibiotic-tolerant persisters. Persister cells are phenotypic variants that exist as a subpopulation within a clonal culture. Being tolerant to lethal antibiotics, they underly the chronic nature of a variety of infections and even help in acquiring genetic resistance. Here, we set out to identify and characterize persistence in S. pneumoniae. Specifically, we followed different strategies to overcome the self-limiting nature of S. pneumoniae as confounding factor in the prolonged monitoring of antibiotic survival needed to study persistence. In optimized conditions, we identified genuine persisters in various growth phases and for four relevant antibiotics through biphasic survival dynamics and heritability assays. Finally, we detected a high variety in antibiotic survival levels across a diverse collection of S. pneumoniae clinical isolates, which shows that a high natural diversity in persistence is widely present in S. pneumoniae. Collectively, this proof-of-concept significantly progresses the understanding of the importance of antibiotic persistence in S. pneumoniae infections which will set stage for characterizing its relevance to clinical outcomes and advocates for increased attention to the phenotype in both fundamental and clinical research. IMPORTANCES. pneumoniae is considered a serious threat by the Centers of Disease Control and Prevention through arising antibiotic resistance. In addition to resistance, bacteria can also survive lethal antibiotic treatment by developing antibiotic tolerance and more specifically by antibiotic tolerance through persistence. This phenotypic variation seems omnipresent among bacterial life, is linked to therapy failure and acts as a catalyst for resistance development. This study gives the first proof of the presence of persister cells in S. pneumoniae and shows a high variety in persistence levels among diverse strains, suggesting persistence is a general trait in S. pneumoniae cultures and that a broad range of genetic elements are controlling the phenotype. Together, our work advocates for higher interest for persistence in S. pneumoniae as a contributing factor for therapy failure and resistance development.