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Original Investigation |

Viability and Virulence of Pneumolysin, Pneumococcal Surface Protein A, and Pneumolysin/Pneumococcal Surface Protein A Mutants in the Ear

Patricia A. Schachern, BS1; Vladimir Tsuprun, PhD1; Sarah Goetz, BA2; Sebahattin Cureoglu, MD1; Steven K. Juhn, MD1; David E. Briles, PhD3; Michael M. Paparella, MD1; Patricia Ferrieri, MD2,4
[+] Author Affiliations
1Department of Otolaryngology, University of Minnesota, Minneapolis
2Department of Pediatrics, University of Minnesota, Minneapolis
3Department of Microbiology, The University of Alabama at Birmingham
4Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis
JAMA Otolaryngol Head Neck Surg. 2013;139(9):937-943. doi:10.1001/jamaoto.2013.4104.
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Importance  Understanding how pneumococcal proteins affect the pathology of the middle ear and inner ear is important for the development of new approaches to prevent otitis media and its complications.

Objectives  To determine the viability and virulence of Streptococcus pneumoniae mutants deficient in pneumolysin (Ply) and pneumococcal surface protein A (PspA) in the chinchilla middle ear.

Design  Bullae of chinchillas were inoculated bilaterally with wild-type (Wt), Ply, PspA, and Ply/PspA strains. Bacterial colony-forming units (CFUs) in middle ear effusions were counted at 48 hours. The CFUs of the PspA group were also counted at 6 to 36 hours after inoculation. Temporal bone histopathological results were compared.

Setting and Participants  Twenty-seven chinchillas in an academic research laboratory.

Exposure  Chinchilla middle ears were inoculated with S pneumoniae to produce sufficient volumes of effusions and noticeable histopathological changes in the ears.

Main Outcomes and Measures  The CFU counts in the middle ear effusions and histopathological changes were compared to determine the effect of pneumococcal protein mutations on chinchilla ears.

Results  At 48 hours, CFUs in middle ears were increased for the Wt and Ply/PspA strains, but Ply remained near inoculum level. No bacteria were detected in the PspA group. The CFUs of PspA decreased over time to a low level at 30 to 36 hours. In vitro, PspA in Todd-Hewitt broth showed an increase in bacterial growth of 2 logs at 43 hours, indicating PspA susceptibility to host defenses in vivo. The PspA and Ply groups had fewer pathologic findings than the Wt or Ply/PspA groups. Histopathological analysis showed significant differences in the number of bacteria in the scala tympani in the Wt group compared with the Ply, PspA, and Ply/PspA groups. The PspA strain was the least virulent.

Conclusions and Relevance  The PspA mutant was much less viable and less virulent in the ear than the Wt, Ply, and Ply/PspA strains. There was no significant attenuation in the viability and virulence of the Ply/PspA mutant compared with the Wt or single mutants. The viability and virulence of pneumococcal mutants seemed to be protein and organ specific.

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Figure 1.
Effects of Mutations in Pneumococcal Surface Protein A (PspA) and Pneumolysin (Ply) on Bacterial Growth

A, 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.

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Figure 2.
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 × 106 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.

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Figure 3.
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.

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