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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.

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Presentation on theme: "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."— Presentation transcript:

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 2 nd most common infectious disease in the first year of life – most common reason for the use of antibiotics in this age group. 2 nd 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. 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.). 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.). 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 organisms in OME cultures (Bluestone et al.). Increased rates of resistant nasopharyngeal isolates in children attending day care (MMWR). 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. 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.” Process occurs via “quorum sensing.”

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6 Importance of Biofilms In-Vivo Biofilms result in chronic inflammation causing collateral damage in addition to direct microbial insults. Biofilms result in chronic inflammation causing collateral damage in addition to direct microbial insults. Biofilms afford microbes protection against opsonization and phagocytosis. Biofilms afford microbes protection against opsonization and phagocytosis. Biofilms render organisms resistant to antimicrobial therapy. 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. Chinchilla model utilized to develop experimental AOM with non-typeable H. influenzae. Biofilm formation demonstrated within 24 hours. Biofilm formation demonstrated within 24 hours. Biofilms also identified on tubes removed in patients with refractory otorrhea post- tympanostomy tube placement. Biofilms also identified on tubes removed in patients with refractory otorrhea post- tympanostomy tube placement. Biofilms identified CLM middle ear biopsies patients with OME 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. Adenoidectomy has been shown to be of benefit in children with RAOM. This benefit remains independently of the size of the adenoid. 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. 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. 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 ? What role do nasopharyngeal biofilms play in the pathogenesis of RAOM ? Are biofilms present on the nasopharyngeal mucosa of otitis prone children ? Are biofilms present on the nasopharyngeal mucosa of otitis prone children ? Do these nasopharyngeal biofilms contain middle ear pathogens ? Do these nasopharyngeal biofilms contain middle ear pathogens ? Do these nasopharyngeal biofilms act as a significant reservoir of 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 N=68, 41 male and 27 female Age range : 3 months to 15 years Age range : 3 months to 15 years Tympanastomy tube (TT) (re)placement + adenoidectomy was performed on all RAOM patients (n=34) Tympanastomy tube (TT) (re)placement + adenoidectomy was performed on all RAOM patients (n=34) Adenoidectomy +/- tonsillectomy was performed on all OSA patients (n=34) Adenoidectomy +/- tonsillectomy was performed on all OSA patients (n=34) IRB approved by Wayne State University IRB approved by Wayne State University

14 Scanning Electron Microscopy of Adenoid Biofilms in RAOM 500x2000x

15 Scanning Electron Microscopy of Adenoid Devoid of Biofilms in OSA Patients 500x2000x Specimen Ad6

16 Adenoid Biofilm Surface Density RAOM vs. OSA

17 Results RAOM patients had an average of 93.53% biofilm coverage RAOM patients had an average of 93.53% biofilm coverage OSA patients had an average of 1.01% biofilm coverage OSA patients had an average of 1.01% biofilm coverage Statistically significant (p<0.0001) using Wilcoxen two sample test 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 Able to present a breakdown of biofilm species Allows images of cells within the matrix Allows images of cells within the matrix Localizes intracellular and extracellular pathogens Localizes intracellular and extracellular pathogens /spring2003/Baxter/MolecularTool.html

19 FISH Probes of Adenoid Specimens with RAOM

20 Makeup of Adenoid Biofilms *Clusters/mm 2

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

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

23 Quantification of Pathogens Because of the sessile nature of organisms in biofilms a true quantification of the pathogenic population is elusive 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 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 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 suggest a method for obtaining clinical data relating to biofilm infections To further establish the validity of RAOM as a biofilm related infectious process 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) 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 Male: 4; Females: 4 Age range: y.o. Age range: y.o. Average age: 6.88 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 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 Tissue digestion was performed on samples overnight to ensure completion

27 Primers Forward and reverse custom DNA primers were obtained for Forward and reverse custom DNA primers were obtained for Human: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) Human: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) S. pneumoniae S. pneumoniae H. influenzae H. influenzae M. catarrhalis 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 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 Analyses were calculated for each bacterial primer set using the following formula 2 (Ct OSA(Bacterial) - Ct RAOM(Bacterial) ) __________________________ 2 (Ct OSA(GAPDH) - Ct RAOM(GAPDH) ) = Corrected Relative Quantity

29 Real Time PCR Yields

30 Results Overall average fold increase for all bacterial species = Overall average fold increase for all bacterial species = Average fold increase by selecting the highest bacterial isolate from each matched pair = 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 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 ? What role do nasopharyngeal biofilms play in the pathogenesis of RAOM ? Are biofilms present on the nasopharyngeal mucosa of otitis prone children ? 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 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 ? Do these nasopharyngeal biofilms contain middle ear pathogens ? FISH imaging demonstrated numerous middle ear pathogens on these nasopharyngeal biofilms 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 ? 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 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 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 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 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 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 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 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 Biofilms are bacterial pathogens that organize in several chronic and recalcitrant infectious processes

39 Study Design Inclusion criteria: pediatric patients with documented CRS or OSA by 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 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 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) Senior author present during imaging with SEM (JEOL JSM-6400) Low (500x), mid (1000x) and high (2000x) resolution pictures were taken of each specimen 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 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 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

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44 Results CRS patients had an average of 92.1% biofilm coverage (range %) 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%) 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 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 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. 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 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 Inflammation of NP URI/GERD/Allergy Inflammation of NP URI/GERD/Allergy NP Colonization by CRS Pathogens NP Colonization by CRS Pathogens NP Biofilm Formation Shedding of Planktonic Organisms Colonization & biofilm formation of maxillary sinus Inflammation of OMC Impaired mucociliary clearance Edema Inflammation of OMC Impaired mucociliary clearance Edema Entrance of CRS pathogens into maxillary sinus Clinical Symptoms Short-Term Oral Antibiotics Shedding of Resistant Sinus Pathogens Shedding of Resistant Sinus Pathogens Interval Improvement

47 Treatment Strategies Chronic Infection CRS Adenoidectomy Maxillary Sinus Aspiration IV Abx Adenoidectomy Limited FESS Adenoidectomy Maxillary Sinus Aspiration 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) 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 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 Mechanical debridement of the adenoid pad may explain the improvement of symptoms in these patients

49 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. Future Directions

50 SUMMARY Dense nasopharyngeal biofilms identified children RAOM Dense nasopharyngeal biofilms identified children RAOM These biofilms contain numerous middle ear pathogens These biofilms contain numerous middle ear pathogens 2000 X quantitative difference middle ear pathogens children RAOM vs OSA 2000 X quantitative difference middle ear pathogens children RAOM vs OSA Nasopharynx of childlren with chronic rhinosinusitis also found to have dense biofilm formations 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 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 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 Mechanical debridement of NP biofilms may explain clinical benefit observed with adenoidectomy

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

53 Research Team James M. Coticchia, M.D. 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.


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