1. Role of Nasopharyngeal Biofilms in Recurrent Acute Otitis Media and Chronic Rhinosinusitis
James M. Coticchia M.D.,F.A.C.S.
Director of Pediatric Otolaryngology
Associate Professor
Vice Chairman
Otolaryngology Head and Neck Surgery
Wayne State University
School of Medicine

2. Prevalence of AOM
2nd most common infectious disease in the first year of life – most common reason for the use of antibiotics in this age group.
5 billion dollars spent on AOM annually in the United States.
68% increase in incidence from the 1970s to the 1990s (Joki-Erkkila et al.).

3. Increase in Resistance and Complications
Increase in suppurative complications of AOM such as mastoiditis (Antonelli et al.).
Increased rates of resistant organisms in OME cultures (Bluestone et al.).
Increased rates of resistant nasopharyngeal isolates in children attending day care (MMWR).

4. Bacterial Biofilms - Description
Proximity of cells to a surface stimulates production of exopolysaccharide matrix – microchannels form connections among microbes and allow continual shedding of planktonic microorganisms.
Process occurs via “quorum sensing.”

6. Importance of Biofilms In-Vivo
Biofilms result in chronic inflammation causing collateral damage in addition to direct microbial insults.
Biofilms afford microbes protection against opsonization and phagocytosis.
Biofilms render organisms resistant to antimicrobial therapy.

7. Previous Identification of Biofilms in OME
Chinchilla model utilized to develop experimental AOM with non-typeable H. influenzae.
Biofilm formation demonstrated within 24 hours.
Biofilms also identified on tubes removed in patients with refractory otorrhea post-tympanostomy tube placement.
Biofilms identified CLM middle ear biopsies patients with OME

8. Adenoidectomy and RAOM
Adenoidectomy has been shown to be of benefit in children with RAOM.
This benefit remains independently of the size of the adenoid.
The mechanism of this benefit is likely related to eradication of nasopharyngeal colonization with middle ear pathogens.

9. Specific Aim
To determine if nasopharyngeal Biofilms play a role in the pathogenesis of RAOM in children.

10. Questions
What role do nasopharyngeal biofilms play in the pathogenesis of RAOM ?
Are biofilms present on the nasopharyngeal mucosa of otitis prone children ?
Do these nasopharyngeal biofilms contain middle ear pathogens ?
Do these nasopharyngeal biofilms act as a significant reservoir of middle ear pathogens ?

11. Methodology Overview Three levels of evaluation Direct visualization
Scanning Electron Microscopy
Qualitative evaluation
Fluorescence In-Situ Hybridization
Quantitative evaluation
Real Time Polymerase Chain Reaction

12. Advantages of Evaluating Biofilms with SEM
Visual representation of the biofilm matrix and its relationship to physiologic structures
Ability to show surface architecture
Evaluates the depth and density of biofilm coverage

13. Study Design N=68, 41 male and 27 female
Age range : 3 months to 15 years
Tympanastomy tube (TT) (re)placement + adenoidectomy was performed on all RAOM patients (n=34)
Adenoidectomy +/- tonsillectomy was performed on all OSA patients (n=34)
IRB approved by Wayne State University
All adenoidectomies performed via curettage

14. Scanning Electron Microscopy of Adenoid Biofilms in RAOM
500x
2000x

15. Scanning Electron Microscopy of Adenoid Devoid of Biofilms in OSA Patients
Specimen Ad6
Biofilm negative specimen. ? Clinical diagnosis error
500x
2000x

16. Adenoid Biofilm Surface Density RAOM vs. OSA

17. Results RAOM patients had an average of 93.53% biofilm coverage
OSA patients had an average of 1.01% biofilm coverage
Statistically significant (p<0.0001) using Wilcoxen two sample test

18. Advantages of Evaluating Biofilms with FISH
Able to present a breakdown of biofilm species
Allows images of cells within the matrix
Localizes intracellular and extracellular pathogens

19. FISH Probes of Adenoid Specimens with RAOM

20. Makeup of Adenoid Biofilms
*Clusters/mm2

21. FISH Photo-Micrographs
10 um
Extracellular biofilms with flourophores
labeling Staphylococcus species. The
bottom strip is a sagittal cut through this slide
Biofilm pods containing S. pneumoniae cells

22. Advantages of Evaluating Biofilms with Real Time Polymerase Chain Reaction
Specific for organism
Very sensitive
Quantitative

23. Quantification of Pathogens
Because of the sessile nature of organisms in biofilms a true quantification of the pathogenic population is elusive
Real time PCR represents a sensitive and well documented technique for quantifying these pathogens through amplification of their unique DNA sequences

24. Project Goals
To create a reproducible method of quantifying bacterial populations in a biofilm matrix on adenoid mucosa
To suggest a method for obtaining clinical data relating to biofilm infections
To further establish the validity of RAOM as a biofilm related infectious process

25. Subjects
Children treated with adenoidectomy for either OSA, or RAOM (4 episodes of acute OM in 6 months, or 6 episodes in 12 months)
Male: 4; Females: 4
Age range: y.o.
Average age: 6.88 y.o.

26. DNA Extraction and Preparation
DNA from each adenoid sample is prepared and purified using the Qiagen QIAmp® Mini Kit using the provided tissue protocol
Tissue digestion was performed on samples overnight to ensure completion

27. Primers Forward and reverse custom DNA primers were obtained for
Human: glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
S. pneumoniae
H. influenzae
M. catarrhalis

28. Real-time Polymerase Chain Reaction (PCR) and Analysis
Three 20μL samples for each specimen were run for each RAOM and OSA specimen
Analyses were calculated for each bacterial primer set using the following formula
(Ct OSA(Bacterial) - Ct RAOM(Bacterial)) 2
Corrected Relative Quantity
__________________________ =
(Ct OSA(GAPDH) - Ct RAOM(GAPDH)) 2

29. Real Time PCR Yields

30. Results
Overall average fold increase for all bacterial species =
Average fold increase by selecting the highest bacterial isolate from each matched pair =
Paired t-test analysis comparing end point Real Time PCR numbers for all bacterial species yielded significance at p=0.045

31. Planktonic Shedding by Nasopharyngeal Biofilms

32. OTITIS PRONE PATHOGENESIS
Entry ME pathogen in nasopharynx ↓
Colonization ME pathogen in nasopharynx
Biofilm production ME pathogen in nasopharynx
Shedding ME pathogen
ET dysfunction
Entry Middle Ear pathogen tubotympanum
Development AOM early Biofilm production ME Pathogen
Antibiotics
Nasopharynx Biofilm Resistance

33. Shedding ME pathogen nasopharynx Biofilm ↓ ET dysfunction
Entry middle ear pathogen tubotympanum
Prophylaxis Tympanostomy Tubes Myringotomy Plus
Adenoidectomy
Decrease Shedding Debride Bioflm ME Debride Biofilm ME
Debride Biofilm Nasopharynx

34. Conclusions
What role do nasopharyngeal biofilms play in the pathogenesis of RAOM ?
Are biofilms present on the nasopharyngeal mucosa of otitis prone children ?
All patients with RAOM had high densities of Biofilms in their nasopharynx (NP), as compared to control specimens

35. Conclusions
Do these nasopharyngeal biofilms contain middle ear pathogens ?
FISH imaging demonstrated numerous middle ear pathogens on these nasopharyngeal biofilms

36. Conclusions
Do these nasopharyngeal biofilms act as a significant reservoir of middle ear pathogens ?
RT-PCR demonstrated 2,059 fold difference of nasopharyngeal middle ear pathogens for RAOM vs. hypertrophy, p<0.05
Biofilm formation by known ME pathogens in the nasopharynx has been clearly demonstrated by these techniques
Resistant biofilms may act as a reservoir for recurrent infections of the tubotympanum
Recent imaging and molecular data such as these challenge the concept of the sterile middle ear effusion

37. BIOFILM DENSITY IN THE PEDIATRIC NASOPHARYNX: CHRONIC RHINOSINUSITIS VERSUS OBSTRUCTIVE SLEEP APNEA

38. Chronic Rhinosinusitis and Biofilms
Pediatric chronic rhinosinusitis (CRS) is a complex disease whose natural history and pathogenesis are poorly understood
CRS can often be recalcitrant even after optimal medical therapy with both oral and IV antibiotics
Recent studies by the CDC estimate that 65% of all human infectious processes involve biofilms
Biofilms are bacterial pathogens that organize in several chronic and recalcitrant infectious processes
Indwelling catheter related sepsis, prostatitis, periodontitis, osteomyelitis, pneumonia, and otitis media
Potera et al. 99% of all bacteria exist in biofilms, and only 1% live in a free floating or planktonic state

39. Study Design
Inclusion criteria: pediatric patients with documented CRS or OSA by
Sinonasal symptoms of at least 12 weeks duration with a failure to respond to a minimum 3-4 week course of a [beta]-lactamase stable antibiotic
Documented OSA with adenoid hypertrophy (+/- tonsillar hypertrophy) and the absence of antibiotic treated tonsillitis/pharyngitis in the past 6 months

40. Experimental Methodology
Senior author present during imaging with SEM (JEOL JSM-6400)
Low (500x), mid (1000x) and high (2000x) resolution pictures were taken of each specimen
Biofilm architecture consistent with the extant literature was easily distinguishable from the barren surface of regions devoid of biofilms
SEM images analyzed using Carnoy software to determine % biofilm coverage of the adenoid mucosal surfaces

41. Low power (500x) SEM image of biofilm architecture from a child with CRS
Low power (500x) SEM image of a barren adenoid surface taken from a patient with OSA

42. High power (2000x) SEM image of biofilm architecture from a child with CRS
High power (2000x) SEM image of a barren adenoid surface taken from a patient with OSA

44. Results
CRS patients had an average of 92.1% biofilm coverage (range %)
OSA patients had an average of 1.1% biofilm coverage (range 0-6.5%)
Statistically significant (p<.001) using non-parametric Mann-Wilcoxon test between the 2 groups

45. Discussion
Adenoid specimens from the CRS group had the majority of their mucosal surfaces covered with biofilms while those in the OSA group were mostly barren
Adenoidectomy has been shown to ameliorate the symptoms of CRS.
The presence of nasopharyngeal biofilms and their embolic shedding of planktonic organisms can thus explain the recalcitrant infections of the maxillary paranasal sinus mucosa despite maximum medical therapy

46. of Resistant Sinus Pathogens Short-Term Oral Antibiotics
Inflammation
of NP
URI/GERD/Allergy
NP Colonization
by CRS Pathogens
NP Biofilm Formation
Entrance of CRS pathogens into maxillary sinus
Inflammation of OMC
Impaired mucociliary clearance Edema
Shedding of Planktonic Organisms
Clinical Symptoms
Colonization & biofilm formation of maxillary sinus
Shedding
of Resistant Sinus Pathogens
Short-Term Oral Antibiotics
Interval Improvement

47. Treatment Strategies Chronic Infection CRS Adenoidectomy
Maxillary Sinus
IV Abx
Aspiration
Limited FESS
Long term oral Abx

48. Conclusion
Biofilms provide a nidus for chronic infection in the pediatric nasopharynx which may seed other areas (paranasal simuses, middle ear)
The reduced susceptibility of biofilms to standard long course oral antibiotics explain the recalcitrant nature of CRS
Mechanical debridement of the adenoid pad may explain the improvement of symptoms in these patients

49. Future Directions Animal Model
To determine the role of bacterial microfilms in the cause of middle ear infections. The animal model to be used will be the chinchilla, which has a similarity to humans in terms of anatomical and neurological structures.

50. SUMMARY Dense nasopharyngeal biofilms identified children RAOM
These biofilms contain numerous middle ear pathogens
2000 X quantitative difference middle ear pathogens children RAOM vs OSA
Nasopharynx of childlren with chronic rhinosinusitis also found to have dense biofilm formations

51. SUMMARY
Nasopharygeal biofilms likely to play an important role in the pathogenesis of recurrent ear infections and chronic rhinosinusits in children
The persistance of biofilm infections may explain the recalcitrant nature of these disease states
Mechanical debridement of NP biofilms may explain clinical benefit observed with adenoidectomy

52. Funding Support The Deafness Research Foundation
Children’s Research Center of Michigan
FMRE
Department of Otolaryngology
Lions Hearing Center of Michigan

53. Research Team James M. Coticchia, M.D. Richard Berk, Ph.D.
Zhong Dong, M.D., Ph.D.
Michael Haupert, D.O.
Abhishek Prasad, M.D.
Giancarlo Zuliani, M.D.
Michael Carron, M.D.
Aaron Duberstein, M.D.
Michael Hoa, M.D.
Livjot Sachdeva, M.S.
Michael Carlisle, B.S.