Antibiotic Tolerance Indicative of Persistence Is Pervasive among Clinical Streptococcus pneumoniae Isolates and Shows Strong Condition Dependence.

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 for S. pneumoniae, little is known about antibiotic-tolerant persisters and robust experimental means are lacking. 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. In this study, 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 a confounding factor in the prolonged monitoring of antibiotic survival needed to study persistence. Under 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 assumes 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 the stage for characterizing its relevance to clinical outcomes and advocates for increased attention to the phenotype in both fundamental and clinical research. IMPORTANCE S. pneumoniae is considered a serious threat by the Centers for Disease Control and Prevention because of rising antibiotic resistance. In addition to resistance, bacteria can also survive lethal antibiotic treatment by developing antibiotic tolerance, more specifically, 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 that persistence is a general trait in S. pneumoniae cultures. Our work advocates for higher interest for persistence in S. pneumoniae as a contributing factor for therapy failure and resistance development.

Neuroblastoma Invasion Strategies Are Regulated by the Extracellular Matrix.

Neuroblastoma is a paediatric malignancy of the developing sympathetic nervous system. About half of the patients have metastatic disease at the time of diagnosis and a survival rate of less than 50%. Our understanding of the cellular processes promoting neuroblastoma metastases will be facilitated by the development of appropriate experimental models. In this study, we aimed to explore the invasion of neuroblastoma cells and organoids from patient-derived xenografts (PDXs) grown embedded in 3D extracellular matrix (ECM) hydrogels by time-lapse microscopy and quantitative image analysis. We found that the ECM composition influenced the growth, viability and local invasion of organoids. The ECM compositions induced distinct cell behaviours, with Matrigel being the preferred substratum for local organoid invasion. Organoid invasion was cell line- and PDX-dependent. We identified six distinct phenotypes in PDX-derived organoids. In contrast, NB cell lines were more phenotypically restricted in their invasion strategies, as organoids isolated from cell line-derived xenografts displayed a broader range of phenotypes compared to clonal cell line clusters. The addition of FBS and bFGF induced more aggressive cell behaviour and a broader range of phenotypes. In contrast, the repression of the prognostic neuroblastoma marker, MYCN, resulted in less aggressive cell behaviour. The combination of PDX organoids, real-time imaging and the novel 3D culture assays developed herein will enable rapid progress in elucidating the molecular mechanisms that control neuroblastoma invasion.

In vitro and in vivo Evaluation of in silico Predicted Pneumococcal UDPG:PP Inhibitors.

Pneumonia, of which Streptococcus pneumoniae is the most common causative agent, is considered one of the three top leading causes of death worldwide. As seen in other bacterial species, antimicrobial resistance is on the rise for this pathogen. Therefore, there is a pressing need for novel antimicrobial strategies to combat these infections. Recently, uridine diphosphate glucose pyrophosphorylase (UDPG:PP) has been put forward as a potential drug target worth investigating. Moreover, earlier research demonstrated that streptococci lacking a functional galU gene (encoding for UDPG:PP) were characterized by significantly reduced in vitro and in vivo virulence. Therefore, in this study we evaluated the anti-virulence activity of potential UDPG:PP inhibitors. They were selected in silico using a tailor-made streptococcal homology model, based on earlier listerial research. While the compounds didn't affect bacterial growth, nor affected in vitro adhesion to and phagocytosis in macrophages, the amount of polysaccharide capsule was significantly reduced after co-incubation with these inhibitors. Moreover, co-incubation proved to have a positive effect on survival in an in vivo Galleria mellonella larval infection model. Therefore, rather than targeting bacterial survival directly, these compounds proved to have an effect on streptococcal virulence by lowering the amount of polysaccharide and thereby probably boosting recognition of this pathogen by the innate immune system. While the compounds need adaptation to broaden their activity to more streptococcal strains rather than being strain-specific, this study consolidates UDPG:PP as a potential novel drug target.