Synergistic inhibition of pneumococcal growth by <i>Dolosigranulum pigrum</i> and <i>Corynebacterium pseudodiphtheriticum:</i> insights into nasopharyngeal microbial interactions

<i>Streptococcus pneumoniae</i> is a nasopharynx colonizer that can invade sterile tissues, causing invasive pneumococcal disease (IPD). <i>Dolosigranulum pigrum</i> and <i>Corynebacterium pseudodiphtheriticum</i> are commensal bacteria commonly isolated from the nasopharynx of healthy children, potentially playing a protective role. This study aims to analyze the effects of <i>D. pigrum</i> and <i>C. pseudodiphtheriticum</i> on <i>S. pneumoniae in vitro</i> growth. Pneumococcal strains were collected from IPD patients and healthy carriers in Catalonia (2016-2023). <i>D. pigrum</i> and <i>C. pseudodiphtheriticum</i> strains were isolated from a healthy child's nasopharynx. <i>S. pneumoniae</i> was co-cultured with each commensal bacterium in triplicate experiments. Pneumococcal growth was quantified using a real-time PCR assay targeting the <i>lytA</i> gene. The effect of commensal bacteria on pneumococcal growth was evaluated using a linear mixed-effect regression model. Twenty-eight pneumococcal strains expressing 24 different serotypes and 27 clonal types were analyzed (18 isolated in blood and 10 in nasopharyngeal aspirate). Pneumococcal growth was decreased by <i>D. pigrum</i> (β = -0.763, 95% confidence interval [CI]: -0.94 to -0.59, <i>P</i> < 0.0001) and <i>C. pseudodiphtheriticum</i> (β = -0.583, 95% CI: -0.76 to -0.41, <i>P</i> < 0.0001). The combined presence of both had a stronger inhibitory effect (β = -0.971, 95% CI: -1.15 to -0.79, <i>P</i> < 0.0001). No association was found between isolation site or serotype with pneumococcal growth. <i>D. pigrum</i> and <i>C. pseudodiphtheriticum</i> significantly reduced pneumococcal growth, with a synergistic effect when combined. This antagonistic effect supports the potential protective factor of healthy nasopharyngeal microbiota against IPD and the development of these microorganisms as probiotics.IMPORTANCEInvasive pneumococcal disease (IPD) is a significant worldwide health challenge. The present study highlights the significant inhibitory effect of two commensal bacteria, <i>Dolosigranulum pigrum</i> and <i>Corynebacterium pseudodiphtheriticum</i>, on pneumococcal growth, with a stronger effect observed when both bacteria are present together. Through testing different strains of <i>S. pneumoniae</i> and the implementation of a robust statistical model, this research advances in the knowledge of microbial ecology and provides evidence to support the development of the use of these commensal bacteria as probiotics. These results emphasize the possibility of using the nasopharyngeal microbiota's natural interactions to mitigate the risk of IPD.