Abstract
Purpose
The aim of the study was to compare the extent of biofilm infection in percentage of mucosal surface area of adenoids removed from children with otitis media with effusion (OME) vs those with recurrent acute otitis media (RAOM) and obstructive sleep apnea (OSA).
Materials and methods
Comparative microanatomical investigation of adenoid mucosa using scanning electron microscopy obtained from 30 children with OME, RAOM, and OSA was used in this study. Seventeen males and 13 females ranging in age from 9 months to 10 years were included in this study. Percentage of biofilm surface area involvement was the main measure.
Results
Adenoids removed from patients with OME had moderately dense mature biofilms covering the mucosal surface with a mean of 27.7% of their mucosal surface covered with mature biofilms. These results were distinct from results obtained from patients diagnosed with RAOM and OSA with means of 97.6% and 0.10% of their mucosal surfaces covered with mature biofilms, respectively. These differences were statistically significant at P < .0001.
Conclusions
Adenoids removed from patients with OME were characterized by distinctly different percentage of biofilm mucosal surface area coverage, with significantly more biofilm presence than OSA patients but significantly less biofilm presence than RAOM patients. Although previous investigations have supported a dominant role of nasopharyngeal biofilms in RAOM pathogenesis, these results suggest nasopharyngeal biofilms may play a different role in the pathogenesis of OME and that this clinical entity may be more multifactorial in nature.
1
Introduction
Otitis media is traditionally recognized as an infectious entity caused by Haemophilus influenzae , Streptococcus pneumoniae , or Moraxella catarrhalis . However, otitis media can be further delineated into 2 subcategories—acute otitis media (AOM) and otitis media with effusion (OME). Clinically, the 2 forms of otitis media present quite differently. Although fluid may be present in the middle ear for both, AOM has a rapid onset with symptoms of otalgia, otorrhea, and fever . Conversely, OME patients present with middle ear fluid but show no signs or symptoms of acute infection and can become chronic (COME) if the effusion persists greater than 3 months . Risk factors for both AOM and OME involve eustachian tube dysfunction, crowded living conditions, day care attendance, and impaired immunologic status .
Moreover, AOM is primarily regarded as a transient infectious entity often with viral coinfection . In contrast, the etiology of otitis media with effusion is hypothesized to be a more protracted but less virulent condition. Initially, OME was thought of as a sterile inflammatory process due to the subsequent inability to culture bacteria from middle ear effusions . It was elucidated that the persistence of proinflammatory cytokines played a key role in the initiation and perpetuation of inflammation . However, cytokines alone do not account for several recent observations regarding OME. Further investigation has also led to the discovery that these sterile effusions have been shown to harbor pathogenic bacterial DNA . In addition, this DNA may be present in effusions for up to 4 weeks after antibiotic therapy and is often concurrently present with bacterial mRNA and proteins —indicating the retained ability for bacterial metabolism. This concept of persistent or recurrent infection in the setting of negative cultures, despite documented bacterial inhabitance, has set the stage for increasing support toward the role of bacterial biofilms in the pathogenesis of otitis media .
Biofilms are complex organized communities of metabolically quiescent bacteria embedded in a matrix that is rich in extracellular polymeric substances . Biofilms initially form as a result of attachment and aggregation to a biological surface and then mature and expand to their final complex structure . It is their unique structure and metabolic properties that confer their resistance to common antibiotics, mechanical shear, radiation, and heat. Principally, antibiotics tend to only be effective toward planktonic or freely swimming bacteria seen on the periphery of the biofilm, whereas the central more metabolically somnolent members of the biofilm remain unscathed, reserving the potential to exhibit planktonic shedding and subsequent infection at a later time . Planktonic shedding is a form of dispersal that occurs through mechanical fragmentation of the biofilm or through the release of single cells induced by cellular signaling . These cells can then move to other areas of the respiratory tract to cause infection or form new biofilms. Thus, biofilms are not only unique in their structure but also unique in their composition.
The effectiveness of adenoidectomies may provide the link between biofilms and their near ubiquitous presence in COME by serving as a bacterial reservoir . A similar link was recently investigated by our laboratory by measuring the biofilm density on adenoidectomy samples from patients with recurrent otitis media and comparing it to those with obstructive sleep apnea (OSA) . The adenoids of children with recurrent acute otitis media (RAOM) showed a confluence of biofilms nearly covering the entire mucosal surface. This was in contrast to the adenoids of children with OSA who exhibited no biofilm formation. In addition, in 2006, Hall-Stoodley and colleagues were the first to demonstrate the direct presence of middle ear mucosal biofilms in children with chronic otitis media. Given that biofilm-colonized adenoids may be a potential source of planktonic bacteria that have the ability to migrate; it reasonable to hypothesize that nasopharyngeal biofilms can disseminate and disperse to the form biofilms in the middle ear thereby causing COME. Thus, we build on these foundations by investigating the presence and role of biofilms on adenoid mucosa in children with COME and compare them to adenoid biofilms in children with both RAOM and OSA.
2
Methods
2.1
Sample collection
Ten samples were obtained from each of the 3 patient groups (OME, RAOM, and OSA). Patients ranged in age from 9 months to 10 years with a mean age of 4.85 years. Recurrent acute otitis media was defined as greater than or equal to 4 episodes of AOM in 6 months or greater than or equal to 6 episodes in 12 months. Patients with OME had evidence of OME greater than or equal to 3 months with no recent history of AOM or antibiotics. Presence of middle ear effusion was documented by both pneumatic otoscopy and tympanometry before surgical procedure. Tympanostomy tube insertion was performed at the time of adenoidectomy to document middle ear findings. All OME patients demonstrated dull, thickened tympanic membranes and mucoid middle ear effusions at the time of tympanostomy tube insertion. Patients with OME who had fluid in their middle ear underwent placement of tympanostomy tubes and collection of middle ear fluid via Juhn Tymp-Tap (Metronic/Xomed, Jacksonville, FL). Furthermore, all RAOM patients demonstrated dull, thickened tympanic membranes and mucopurulent middle ear effusions at the time of tympanostomy tube insertion. All samples were acquired from adenoidectomies performed for OME, RAOM, and OSA, respectively, by the authors at The Children’s Hospital of Michigan (Detroit, MI), St Joseph Mercy Hospital (Pontiac, MI), Lahser Ambulatory Surgery Center (Southfield, MI), and William Beaumont Hospital (Royal Oak, MI). All patients from whom adenoid samples were obtained had the sole diagnosis of OME or RAOM or OSA. Institutional review board approval was attained from Wayne State University (Detroit, MI).
2.2
Scanning electron microscopy preparation
All samples were prepared for scanning electron microscopy (SEM) using the following methodology: tissue samples were fixed for 3 hours in 2.5% glutaraldehyde. Distilled water was used for washing. Four washes were done for 15 minutes each. The samples were then treated with 1% osmium tetroxide for 30 minutes. The tissue was dehydrated with the following ethanol concentrations for 15 minutes each: 30%, 50%, 70%, 90%, 100%, and 100%. Tissue was then washed with hexamethyldisilvan (Electron Microscopy Sciences, Hatfield, PA) 4 times for 15 minutes. Several drops of hexamethyldisilvan were placed on the samples and were left to dry for 48 hours under a hood. Samples were then mounted and gold sputter coated in final preparation for imaging.
2.3
Scanning electron microscopy imaging and analysis
Imaging was performed at the University of Michigan-Ann Arbor and at Wayne State University School of Medicine. The senior author was present at all imaging sessions. Specimens were imaged at 500× and 1500×. Biofilm architecture consistent with the literature was noted and analyzed using the Carnoy image analysis software obtained from the Web site www.kuleuven.ac.be/bio/sys/carnoy . The software was used to quantify the percentage of adenoidal surface are covered by biofilm.
2
Methods
2.1
Sample collection
Ten samples were obtained from each of the 3 patient groups (OME, RAOM, and OSA). Patients ranged in age from 9 months to 10 years with a mean age of 4.85 years. Recurrent acute otitis media was defined as greater than or equal to 4 episodes of AOM in 6 months or greater than or equal to 6 episodes in 12 months. Patients with OME had evidence of OME greater than or equal to 3 months with no recent history of AOM or antibiotics. Presence of middle ear effusion was documented by both pneumatic otoscopy and tympanometry before surgical procedure. Tympanostomy tube insertion was performed at the time of adenoidectomy to document middle ear findings. All OME patients demonstrated dull, thickened tympanic membranes and mucoid middle ear effusions at the time of tympanostomy tube insertion. Patients with OME who had fluid in their middle ear underwent placement of tympanostomy tubes and collection of middle ear fluid via Juhn Tymp-Tap (Metronic/Xomed, Jacksonville, FL). Furthermore, all RAOM patients demonstrated dull, thickened tympanic membranes and mucopurulent middle ear effusions at the time of tympanostomy tube insertion. All samples were acquired from adenoidectomies performed for OME, RAOM, and OSA, respectively, by the authors at The Children’s Hospital of Michigan (Detroit, MI), St Joseph Mercy Hospital (Pontiac, MI), Lahser Ambulatory Surgery Center (Southfield, MI), and William Beaumont Hospital (Royal Oak, MI). All patients from whom adenoid samples were obtained had the sole diagnosis of OME or RAOM or OSA. Institutional review board approval was attained from Wayne State University (Detroit, MI).
2.2
Scanning electron microscopy preparation
All samples were prepared for scanning electron microscopy (SEM) using the following methodology: tissue samples were fixed for 3 hours in 2.5% glutaraldehyde. Distilled water was used for washing. Four washes were done for 15 minutes each. The samples were then treated with 1% osmium tetroxide for 30 minutes. The tissue was dehydrated with the following ethanol concentrations for 15 minutes each: 30%, 50%, 70%, 90%, 100%, and 100%. Tissue was then washed with hexamethyldisilvan (Electron Microscopy Sciences, Hatfield, PA) 4 times for 15 minutes. Several drops of hexamethyldisilvan were placed on the samples and were left to dry for 48 hours under a hood. Samples were then mounted and gold sputter coated in final preparation for imaging.
2.3
Scanning electron microscopy imaging and analysis
Imaging was performed at the University of Michigan-Ann Arbor and at Wayne State University School of Medicine. The senior author was present at all imaging sessions. Specimens were imaged at 500× and 1500×. Biofilm architecture consistent with the literature was noted and analyzed using the Carnoy image analysis software obtained from the Web site www.kuleuven.ac.be/bio/sys/carnoy . The software was used to quantify the percentage of adenoidal surface are covered by biofilm.
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