Emergence of multidrug-resistant ST876 serotype 14 Streptococcus pneumoniae with spontaneous mutations driving pathogenesis: genomic and clinical insights from pneumococcal pericarditis and global screening

Figures

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  Figure 1. The phylogenetic relationship between hz23B and hz23P and bloodstream infection-associated Serotype 14 Streptococcus pneumoniae. (A) cgMLST results of hz23B, hz23P, and all other blood-isolated serotype 14 pneumococcal strains (n=10) in our lab. The lines connecting the circles represent the clonal relationships between different isolates, with the numbers on the lines indicating the number of core gene differences. (B) The heat map shows the number of single-nucleotide polymorphisms (SNPs) between hz23B, hz23P, and all other blood-isolated serotype 14 pneumococcal strains in our lab.

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  Figure 2. The mutation screening of all serotype 14 S. pneumoniae and protein structure prediction and modeling. (A) The phylogenetic tree shows the majority of all tested serotype 14 pneumococci (n=63) belonging to CC876, including ST876 and 4 newly assigned STs (19436, 19504, 19428, and 19519) as marked in the inner cycle of the figure. Only one strain (hz23P) was isolated from the pericardial effusion, which was marked as black on the second cycle. The mutations of CiaH_W192L and FabT_E21A were screened for all strains, and only hz23P carries those marked in the outside two cycles. (B, C) Protein structure prediction and modeling. Ribbon representations of the CiaH and FabT structure. The mutated hz23P (CiaH_W192L) was marked green in the zoomed-in panel. FabT from strain hz23B (gray in the roomed-in panel) was found to identify 100% with an X-ray crystal structure in a complex with DNA (PDB ID: 6JBX). The FabT variant of strain hz23P carrying FabT_E21A was marked in pink in the zoomed-in panel.

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  Figure 3. Adhesion and invasion test on human epithelial cells of hz23B and hz23P. The first panel shows the adhesion test results from both strains on lung cells HBE and human nasopharyngeal cells Detroit562 (A). The second panel shows the translocation test results from both strains of isolates on lung cells HBE and human nasopharyngeal cells Detroit562 (B). The third panel shows the barrier destruction ability of both strains on lung cells HBE and human nasopharyngeal cells Detroit562 (C). * and **** represent a significant difference with a p < 0.05, p < 0.0001, respectively.

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  Figure 4. Survival test under an anaerobic environment. Bacterial counts of four tested strains cultured in rich-nutrition medium THY (A), low-nutrition medium BSA+G1g (B), and high-glucose medium BSA+G2g (C) for 4, 8, and 16 hours. hz23P exhibits a unique growth advantage under low-nutrient (BSA+G1g) and anaerobic conditions. Single-point mutants (hz23B::ciaH_W192L, hz23B::fabT_E21A) display partial or delayed phenotypes, suggesting synergistic interaction of the double mutation. **** and *** represent significant differences with p < 0.0001 and < 0.001, respectively.

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  Figure 5. Global distribution of resistance and pericarditis-related mutations in serotype 14 strains. (A) The world map marked with the distribution of all sequence types (STs) of serotype 14 strains. The size of the circle indicates the number of isolates. The gradient blue shows the global distribution of different STs other than ST876, and red represents the occurrence of ST876; (B) The phylogenetic tree of all 1429 serotype 14 strains annotated with STs, CRO, PEN, and ERY non-susceptible (yellow) and resistant (red), CiaH_W192L/FabT_E21A (black) for each strain.