Longitudinal optical coherence tomography to visualize the in vivo response of middle ear biofilms to antibiotic therapy

Abstract

Studying the impact of antibiotic treatment on otitis media (OM), the leading cause of primary care office visits during childhood, is critical to develop appropriate treatment strategies. Tracking dynamic middle ear conditions during antibiotic treatment is not readily applicable in patients, due to the limited diagnostic techniques available to detect the smaller amount and variation of middle ear effusion (MEE) and middle ear bacterial biofilm, responsible for chronic and recurrent OM. To overcome these challenges, a handheld optical coherence tomography (OCT) system has been developed to monitor in vivo response of biofilms and MEEs in the OM-induced chinchilla model, the standard model for human OM. As a result, the formation of MEE as well as biofilm adherent to the tympanic membrane (TM) was longitudinally assessed as OM developed. Various types of MEEs and biofilms in the chinchilla model were identified, which showed comparable features as those in humans. Furthermore, the effect of antibiotics on the biofilm as well as the amount and type of MEEs was investigated with low-dose and high-dose treatment (ceftriaxone). The capability of OCT to non-invasively track and examine middle ear conditions is highly beneficial for therapeutic OM studies and will lead to improved management of OM in patients.

Figure 1

Handheld OCT system for middle ear imaging in the chinchilla model. (a) Photo of the OCT system and handheld probe (inset). Representative OCT images showing (b) clear middle ear cavity, (c) presence of a MEE, and (d,e) TM with a biofilm. The TM and any biofilm adherent to the TM was segmented. Next, the orthogonal distance from the green and the red line was measured and corrected for the refractive index (RI) error. Scale bars represent 100 µm. TM tympanic membrane, MEC middle ear cavity, MEE middle ear effusion.

Figure 2

OM-induced changes in the middle ear cavity detected using OCT. Representative OCT and otoscopy (inset) images of (a) control chinchilla without induced AOM, and (b) AOM-induced chinchilla during the progression of OM. Yellow arrows indicate a MEE-air boundary, red arrows show scattering particle aggregates suspended in the MEEs, and white arrows indicate biofilm. Blue arrows in otoscopy images indicate white biomass behind the TM. (c) Plot of the averaged TM thickness measured from OCT with standard deviation for a specific animal shown in (a,b). The solid line was determined from the linear polynomial fitting. The corresponding p-value on the fitted slope was determined from a two-sided t test with the null hypothesis of a slope of zero. A positive slope with a p-value less than 0.05 indicates a significant increasing trend in the thickness. (d) Histologic images on day 13 demonstrate distinct differences in the middle ear cavity (MEC) where significant amount of MEE and immune cell infiltration were present in OM-affected bulla, but absent in the control without induced AOM. (e) Confocal laser scanning microscope images from fluorescence in situ hybridization (FISH). A nuclei stain (DAPI) detects unspecified genetic components, whereas the green fluorescence indicates the presence of Haemophilus influenzae on the TM. Scale bars in OCT, histologic images, and FISH represent 100 µm, 3 mm, and 10 µm, respectively.

Figure 3

Formation and growth of a TM-adherent biofilm in vivo. (a) OCT and otoscopy (inset) images showing the formation of the biofilm in an AOM-induced chinchilla. Starting day 9, a cluster of biomass adhered to the TM was observed, which continued to accumulate until day 21 (white arrows). Blue arrows indicate earwax that limited visual assessment of the TM. Scale bars represent 100 µm. (b) The averaged TM thickness and standard deviation were plotted for the control (n = 3) and OM-induced group (n = 10), where n indicates the number of animals. Note that animals longitudinally tracked for longer than day 3 post-inoculation are included in the plot. The thickness measured from the specific animal in (a) is shown in blue. The positive slope from the linear polynomial fitting with a p-value of 0.0015 indicates a significant increasing trend in the thickness. STD standard deviation.

Figure 4

OCT images of different MEE representations in chinchilla compared with those in human subjects. (a) Retracted TM and the presence of a scant MEE. (b) Particulate-like scatterers present in transparent fluid. (c) A dense, highly scattering tissue-like (brighter OCT signals) MEE (blue arrows). (d) The presence of a TM biofilm as well as dense biomass. Images were acquired from different chinchillas. (e) OCT image of a scant MEE and biofilm from a 3-year-old human subject diagnosed with bilateral recurrent acute suppurative OM. Inset figures represent the otoscope surface view of the TM. (f) OCT and otoscope images of a fully filled watery MEE from a 2-year-old subject diagnosed with chronic serous OM. (g) OCT and otoscope images from a 1-year-old subject diagnosed with recurrent acute OM. (h) OCT and otoscope images of biofilm and MEE from a 14-month-old subject diagnosed with recurrent acute OM. All scale bars represent 200 µm.

Figure 5

Clearance of MEE after antibiotic therapy longitudinally observed with OCT. (a) Plot of the averaged TM thickness and standard deviation in the control, OM-induced without antibiotics, OM-induced with low-dose and high-dose antibiotics groups, where n indicates the number of animals. Note that animals longitudinally tracked for longer than day 3 post-inoculation are included in the plot. Plots from the representative animals in (b) and (c) are shown in solid blue and green lines, respectively. OCT and otoscopy (inset) images of AOM-induced chinchillas in (b) low-dose and (c) high-dose antibiotic treatment groups. In (b), OCT revealed the absence of MEE and the presence of an additional scattering layer affixed to the TM (white arrow) on day 13. In (c), a chinchilla receiving high-dose treatment displayed resolution of OM with no MEE detected on and after day 9. Scale bars in OCT represent 100 µm. (d) Histologic images of (b) and (c) on day 13 and day 10, respectively. (e) Confocal laser scanning microscope images from fluorescence in situ hybridization (FISH). A nuclei stain (DAPI) detects unspecified genetic components, whereas the green fluorescence indicates the presence of NTHi on the TM. *Animals in the OM-induced without antibiotics group from Fig. 3b are also included. Three animals in the infected without antibiotics group spontaneously healed and were not included in the plot in (a). STD standard deviation.

Figure 6

The presence of MEE and biofilm remained after antibiotic therapy. OCT and otoscopy (inset) images of two AOM-induced chinchillas from (a) low-dose and (b) high-dose antibiotic treatment groups. Both chinchillas showed the presence of the MEE (red arrows) through day 10, and a thick biofilm was visualized (white arrows). OCT image boundaries in orange note days given antibiotics. Scale bars represent 100 µm. (c) Histologic images of (a) and (b) on day 10, visualizing the MEEs in the middle ear cavities (MECs).