1. Date of download: 6/28/2016 Copyright © 2016 American Medical Association. All rights reserved. From: Viability and Virulence of Pneumolysin, Pneumococcal Surface Protein A, and Pneumolysin/Pneumococcal Surface Protein A Mutants in the Ear JAMA Otolaryngol Head Neck Surg. 2013;139(9):937-943. doi:10.1001/jamaoto.2013.4104 Effects of Mutations in Pneumococcal Surface Protein A (PspA) and Pneumolysin (Ply) on Bacterial GrowthA, At 48 hours after inoculation, colony-forming units (CFUs) in middle ear effusion (MEE) were significantly lower (P <.001) for the Ply-deficient (Ply − ) and PspA-deficient (PspA − ) mutants compared with the wild-type (Wt) and Ply − /PspA − strains. Significant differences (P <.001) in CFU counts were seen between the Ply − and PspA − mutants. Counts for the Wt and Ply − /PspA − strains were increased, while those for Ply − remained near the initial inoculum levels. No CFUs were detected in the PspA − group. B, In MEE, CFUs of the PspA − mutant decreased steadily over time, with few remaining at 30 hours after inoculation. However, after 43 hours in Todd-Hewitt broth (THB), there was an increase of about 2 logs. Figure Legend:

2. Date of download: 6/28/2016 Copyright © 2016 American Medical Association. All rights reserved. From: Viability and Virulence of Pneumolysin, Pneumococcal Surface Protein A, and Pneumolysin/Pneumococcal Surface Protein A Mutants in the Ear JAMA Otolaryngol Head Neck Surg. 2013;139(9):937-943. doi:10.1001/jamaoto.2013.4104 A Comparison of Pathologic Changes of the Round Window Membrane (RWM) and the Scala Tympani (ST)Pathologic changes of the RWM and ST and bacterial counts of middle ear effusions (MEEs) 48 hours after inoculation with approximately 1 × 10 6 colony- forming units (CFUs)/mL of the indicated strains. A, Although not significant, the RWM of the single-mutant groups tended to be less thick than that of the wild-type (Wt) or double-mutant groups. B and C, No significant difference was found among the groups in the number of inflammatory cells; however, the pneumococcal surface protein A–deficient (PspA − ) mutant had the fewest cells in the RWM and the ST. D, A significant difference was observed in the number of free-floating bacteria per area counted by light microscopy in tissue sections stained with toluidine blue in the ST between the Wt and the pneumolysin-deficient (Ply − ) (P =.009), Wt and PspA − (P =.006), and Wt and Ply − /PspA − (P =.03) groups. No bacteria were seen in the PspA − group. E, No significant differences were seen in the numbers of phagocytized bacteria in inflammatory cells in the ST, but fewer were seen in the PspA − group. F, Light microscopic analysis of the ST for the PspA − group found no detectable bacteria at 48 hours, consistent with CFU counts in MEE at 48 hours after infection. Figure Legend:

3. Date of download: 6/28/2016 Copyright © 2016 American Medical Association. All rights reserved. From: Viability and Virulence of Pneumolysin, Pneumococcal Surface Protein A, and Pneumolysin/Pneumococcal Surface Protein A Mutants in the Ear JAMA Otolaryngol Head Neck Surg. 2013;139(9):937-943. doi:10.1001/jamaoto.2013.4104 Histopathological Analysis of Round Window Membranes (RWMs)Arrows show the boundary of the RWM, and arrowheads indicate bacteria (toluidine blue stain, original magnification ×1000). A, This RWM from a chinchilla inoculated with the wild-type (Wt) strain is thickened from inflammatory cell infiltration. Bacteria in the scala tympani (ST) can be seen both free floating and within inflammatory cells. B, After inoculation with the pneumolysin-deficient (Ply − ) mutant, RWM histopathological findings were similar to those of the Wt and double-mutant strains, with infiltration of inflammatory cells and bacteria within the ST. C, The RWM from this animal inoculated with the pneumococcal surface protein A–deficient (PspA − ) mutant was not as thick compared with the other groups and did not show bacterial penetration into the ST. D, No pathologic attenuation was observed in the Ply − /PspA − strain compared with that in the Wt or single-mutant strains. ME indicates middle ear. Figure Legend: