Development of a robust and quantitative high-throughput screening method for assessing phenotypic variation in large Neisseria meningitidis isolate collections.

Genome-wide association studies are a powerful approach for identifying determinants of disease. For infectious diseases, high throughput assays are required for measuring the variance in multiple virulence-related phenotypes of large bacterial isolate collections and for association of this phenotypic variance with genotype. The primary limiting factors are cost, effectiveness and a standardized inoculum. A method was developed to create an inoculum array of multiple isolates that could be used for a series of high-throughput multi-isolate phenotypic investigations in a laboratory setting. A key starting point was the standardisation of the inoculum by production of identical batches of each isolate from cells grown to mid-log phase. Cultures with pre-determined optical densities were aliquoted in set patterns into multiple multi-well plates containing 50% glycerol and stored at -80 °C. Prior to a specific assay, an inoculum plate was defrosted and subjected to a brief period of incubation. Control strains can be placed on each plate in order to control for intra-assay variability. A high throughput screen is described in detail for quantification of biofilm formation. This example utilised the crystal violet staining method and multi-assay stock plates containing 16 meningococcal isolates.•Multi-assay stock plate of exponentially growing isolates is cost-effective and simple to implement in a laboratory setting.•This method would predict realistic standard deviations for multiple isolates in phenotypic assays and generate data for performance of power calculations for genotyping.•This method has the potential to identify both known and unknown genetic determinants of phenotypic variability for each tested isolate when paired with genetic analysis of whole genome sequencing data.

Nasal pneumococcal density is associated with microaspiration and heightened human alveolar macrophage responsiveness to bacterial pathogens

Rationale: Pneumococcal pneumonia remains a global health problem. Colonization of the nasopharynx with Streptococcus pneumoniae (Spn), although a prerequisite of infection, is the main source of exposure and immunological boosting in children and adults. However, our knowledge of how nasal colonization impacts on the lung cells, especially on the predominant alveolar macrophage (AM) population, is limited. Objectives: Using a controlled human infection model to achieve nasal colonization with 6B serotype, we investigated the effect of Spn colonization on lung cells. Methods: We collected BAL from healthy pneumococcal-challenged participants aged 18–49 years. Confocal microscopy and molecular and classical microbiology were used to investigate microaspiration and pneumococcal presence in the lower airways. AM opsonophagocytic capacity was assessed by functional assays in vitro, whereas flow cytometry and transcriptomic analysis were used to assess further changes on the lung cellular populations. Measurements and Main Results: AMs from Spn-colonized individuals exhibited increased opsonophagocytosis to pneumococcus (11.4% median increase) for approximately 3 months after experimental pneumococcal colonization. AMs also had increased responses against other bacterial pathogens. Pneumococcal DNA detected in the BAL samples of Spn-colonized individuals were positively correlated with nasal pneumococcal density (r=0.71; P=0.029). Similarly, AM heightened opsonophagocytic capacity was correlated with nasopharyngeal pneumococcal density (r=0.61, P=0.025). Conclusions: Our findings demonstrate that nasal colonization with pneumococcus and microaspiration prime AMs, leading to brisker responsiveness to both pneumococcus and unrelated bacterial pathogens. The relative abundance of AMs in the alveolar spaces, alongside their potential for nonspecific protection, render them an attractive target for novel vaccines.

Nasal pneumococcal density is associated with microaspiration and heightened human alveolar macrophage responsiveness to bacterial pathogens

Rationale: Pneumococcal pneumonia remains a global health problem. Colonization of the nasopharynx with Streptococcus pneumoniae (Spn), although a prerequisite of infection, is the main source of exposure and immunological boosting in children and adults. However, our knowledge of how nasal colonization impacts on the lung cells, especially on the predominant alveolar macrophage (AM) population, is limited. Objectives: Using a controlled human infection model to achieve nasal colonization with 6B serotype, we investigated the effect of Spn colonization on lung cells. Methods: We collected BAL from healthy pneumococcal-challenged participants aged 18–49 years. Confocal microscopy and molecular and classical microbiology were used to investigate microaspiration and pneumococcal presence in the lower airways. AM opsonophagocytic capacity was assessed by functional assays in vitro, whereas flow cytometry and transcriptomic analysis were used to assess further changes on the lung cellular populations. Measurements and Main Results: AMs from Spn-colonized individuals exhibited increased opsonophagocytosis to pneumococcus (11.4% median increase) for approximately 3 months after experimental pneumococcal colonization. AMs also had increased responses against other bacterial pathogens. Pneumococcal DNA detected in the BAL samples of Spn-colonized individuals were positively correlated with nasal pneumococcal density (r=0.71; P=0.029). Similarly, AM heightened opsonophagocytic capacity was correlated with nasopharyngeal pneumococcal density (r=0.61, P=0.025). Conclusions: Our findings demonstrate that nasal colonization with pneumococcus and microaspiration prime AMs, leading to brisker responsiveness to both pneumococcus and unrelated bacterial pathogens. The relative abundance of AMs in the alveolar spaces, alongside their potential for nonspecific protection, render them an attractive target for novel vaccines.