Otopathogenic Staphylococcus aureus Invades Human Middle Ear Epithelial Cells Primarily through Cholesterol Dependent Pathway.

Chronic suppurative otitis media (CSOM) is one of the most common infectious diseases of the middle ear especially affecting children, leading to delay in language development and communication. Although Staphylococcus aureus is the most common pathogen associated with CSOM, its interaction with middle ear epithelial cells is not well known. In the present study, we observed that otopathogenic S. aureus has the ability to invade human middle ear epithelial cells (HMEECs) in a dose and time dependent manner. Scanning electron microscopy demonstrated time dependent increase in the number of S. aureus on the surface of HMEECs. We observed that otopathogenic S. aureus primarily employs a cholesterol dependent pathway to colonize HMEECs. In agreement with these findings, confocal microscopy showed that S. aureus colocalized with lipid rafts in HMEECs. The results of the present study provide new insights into the pathogenesis of S. aureus induced CSOM. The availability of in vitro cell culture model will pave the way to develop novel effective treatment modalities for CSOM beyond antibiotic therapy.

Influence of different sized files on the accuracy of two electronic apex locators.

The aim of this study was to evaluate the impact of different file sizes on the accuracy of two electronic apex locators (EALs). Thirty extracted human single-rooted permanent mandibular incisors were used. A #10 K-file was inserted in the root canal until its end could be observed (using a light microscope) through the apical foramen. One millimetre was subtracted to establish working length (WL). Electronic readings were performed using MiniApex Locator or Root ZX II, from #10 K-file to #130 K-file. Statistical analysis was performed by two-way anova and Tukey test (P ≤ 0.05). From #60 to #130 K-file, observed differences were noted between the values obtained with both EALs and WL (P ≤ 0.05). The MiniApex Locator showed increased means when measurements were made with #50 to #70 and with #120 (P = 0.008) and #130 (P = 0.005) K-files. File sizes influenced the accuracy of EALs - the greater the instrumentation size, the higher mean differences compared to WL.

Otopathogenic Pseudomonas aeruginosa Enters and Survives Inside Macrophages.

Otitis media (OM) is a broad term describing a group of infectious and inflammatory disorders of the middle ear. Despite antibiotic therapy, acute OM can progress to chronic suppurative otitis media (CSOM) characterized by ear drum perforation and purulent discharge. Pseudomonas aeruginosa is the most common pathogen associated with CSOM. Although, macrophages play an important role in innate immune responses but their role in the pathogenesis of P. aeruginosa-induced CSOM is not known. The objective of this study is to examine the interaction of P. aeruginosa with primary macrophages. We observed that P. aeruginosa enters and multiplies inside human and mouse primary macrophages. This bacterial entry in macrophages requires both microtubule and actin dependent processes. Transmission electron microscopy demonstrated that P. aeruginosa was present in membrane bound vesicles inside macrophages. Interestingly, deletion of oprF expression in P. aeruginosa abrogates its ability to survive inside macrophages. Our results suggest that otopathogenic P. aeruginosa entry and survival inside macrophages is OprF-dependent. The survival of bacteria inside macrophages will lead to evasion of killing and this lack of pathogen clearance by phagocytes contributes to the persistence of infection in CSOM. Understanding host-pathogen interaction will provide novel avenues to design effective treatment modalities against OM.