Presentation on theme: "James M. Coticchia M.D.,F.A.C.S. Director of Pediatric Otolaryngology"— Presentation transcript:
1Role of Nasopharyngeal Biofilms in Recurrent Acute Otitis Media and Chronic Rhinosinusitis James M. Coticchia M.D.,F.A.C.S.Director of Pediatric OtolaryngologyAssociate ProfessorVice ChairmanOtolaryngology Head and Neck SurgeryWayne State UniversitySchool of Medicine
2Prevalence of AOM2nd 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.).
3Increase 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).
4Bacterial 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.”
6Importance 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.
7Previous 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
8Adenoidectomy 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.
9Specific AimTo determine if nasopharyngeal Biofilms play a role in the pathogenesis of RAOM in children.
10QuestionsWhat 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 ?
11Methodology Overview Three levels of evaluation Direct visualization Scanning Electron MicroscopyQualitative evaluationFluorescence In-Situ HybridizationQuantitative evaluationReal Time Polymerase Chain Reaction
12Advantages of Evaluating Biofilms with SEM Visual representation of the biofilm matrix and its relationship to physiologic structuresAbility to show surface architectureEvaluates the depth and density of biofilm coverage
13Study Design N=68, 41 male and 27 female Age range : 3 months to 15 yearsTympanastomy 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 UniversityAll adenoidectomies performed via curettage
14Scanning Electron Microscopy of Adenoid Biofilms in RAOM 500x2000x
15Scanning Electron Microscopy of Adenoid Devoid of Biofilms in OSA Patients Specimen Ad6Biofilm negative specimen. ? Clinical diagnosis error500x2000x
21FISH Photo-Micrographs 10 umExtracellular biofilms with flourophoreslabeling Staphylococcus species. Thebottom strip is a sagittal cut through this slideBiofilm pods containing S. pneumoniae cells
22Advantages of Evaluating Biofilms with Real Time Polymerase Chain Reaction Specific for organismVery sensitiveQuantitative
23Quantification of Pathogens Because of the sessile nature of organisms in biofilms a true quantification of the pathogenic population is elusiveReal time PCR represents a sensitive and well documented technique for quantifying these pathogens through amplification of their unique DNA sequences
24Project GoalsTo create a reproducible method of quantifying bacterial populations in a biofilm matrix on adenoid mucosaTo suggest a method for obtaining clinical data relating to biofilm infectionsTo further establish the validity of RAOM as a biofilm related infectious process
25SubjectsChildren 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: 4Age range: y.o.Average age: 6.88 y.o.
26DNA Extraction and Preparation DNA from each adenoid sample is prepared and purified using the Qiagen QIAmp® Mini Kit using the provided tissue protocolTissue digestion was performed on samples overnight to ensure completion
27Primers Forward and reverse custom DNA primers were obtained for Human: glyceraldehyde-3-phosphate dehydrogenase (GAPDH)S. pneumoniaeH. influenzaeM. catarrhalis
28Real-time Polymerase Chain Reaction (PCR) and Analysis Three 20μL samples for each specimen were run for each RAOM and OSA specimenAnalyses were calculated for each bacterial primer set using the following formula(Ct OSA(Bacterial) - Ct RAOM(Bacterial))2Corrected Relative Quantity__________________________=(Ct OSA(GAPDH) - Ct RAOM(GAPDH))2
30ResultsOverall 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
32OTITIS PRONE PATHOGENESIS Entry ME pathogen in nasopharynx↓Colonization ME pathogen in nasopharynxBiofilm production ME pathogen in nasopharynxShedding ME pathogenET dysfunctionEntry Middle Ear pathogen tubotympanumDevelopment AOM early Biofilm production ME PathogenAntibioticsNasopharynx Biofilm Resistance
33Shedding ME pathogen nasopharynx Biofilm ↓ ET dysfunction Entry middle ear pathogen tubotympanumProphylaxis Tympanostomy Tubes Myringotomy PlusAdenoidectomyDecrease Shedding Debride Bioflm ME Debride Biofilm MEDebride Biofilm Nasopharynx
34ConclusionsWhat 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
35ConclusionsDo these nasopharyngeal biofilms contain middle ear pathogens ?FISH imaging demonstrated numerous middle ear pathogens on these nasopharyngeal biofilms
36ConclusionsDo 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<0.05Biofilm formation by known ME pathogens in the nasopharynx has been clearly demonstrated by these techniquesResistant biofilms may act as a reservoir for recurrent infections of the tubotympanumRecent imaging and molecular data such as these challenge the concept of the sterile middle ear effusion
37BIOFILM DENSITY IN THE PEDIATRIC NASOPHARYNX: CHRONIC RHINOSINUSITIS VERSUS OBSTRUCTIVE SLEEP APNEA
38Chronic Rhinosinusitis and Biofilms Pediatric chronic rhinosinusitis (CRS) is a complex disease whose natural history and pathogenesis are poorly understoodCRS can often be recalcitrant even after optimal medical therapy with both oral and IV antibioticsRecent studies by the CDC estimate that 65% of all human infectious processes involve biofilmsBiofilms are bacterial pathogens that organize in several chronic and recalcitrant infectious processesIndwelling catheter related sepsis, prostatitis, periodontitis, osteomyelitis, pneumonia, and otitis mediaPotera et al. 99% of all bacteria exist in biofilms, and only 1% live in a free floating or planktonic state
39Study DesignInclusion criteria: pediatric patients with documented CRS or OSA bySinonasal symptoms of at least 12 weeks duration with a failure to respond to a minimum 3-4 week course of a [beta]-lactamase stable antibioticDocumented OSA with adenoid hypertrophy (+/- tonsillar hypertrophy) and the absence of antibiotic treated tonsillitis/pharyngitis in the past 6 months
40Experimental Methodology Senior author present during imaging with SEM (JEOL JSM-6400)Low (500x), mid (1000x) and high (2000x) resolution pictures were taken of each specimenBiofilm architecture consistent with the extant literature was easily distinguishable from the barren surface of regions devoid of biofilmsSEM images analyzed using Carnoy software to determine % biofilm coverage of the adenoid mucosal surfaces
41Low 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
42High 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
44ResultsCRS 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
45DiscussionAdenoid specimens from the CRS group had the majority of their mucosal surfaces covered with biofilms while those in the OSA group were mostly barrenAdenoidectomy 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
46of Resistant Sinus Pathogens Short-Term Oral Antibiotics Inflammationof NPURI/GERD/AllergyNP Colonizationby CRS PathogensNP Biofilm FormationEntrance of CRS pathogens into maxillary sinusInflammation of OMCImpaired mucociliary clearance EdemaShedding of Planktonic OrganismsClinical SymptomsColonization & biofilm formation of maxillary sinusSheddingof Resistant Sinus PathogensShort-Term Oral AntibioticsInterval Improvement
48ConclusionBiofilms 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 CRSMechanical debridement of the adenoid pad may explain the improvement of symptoms in these patients
49Future 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.
50SUMMARY Dense nasopharyngeal biofilms identified children RAOM These biofilms contain numerous middle ear pathogens2000 X quantitative difference middle ear pathogens children RAOM vs OSANasopharynx of childlren with chronic rhinosinusitis also found to have dense biofilm formations
51SUMMARYNasopharygeal biofilms likely to play an important role in the pathogenesis of recurrent ear infections and chronic rhinosinusits in childrenThe persistance of biofilm infections may explain the recalcitrant nature of these disease statesMechanical debridement of NP biofilms may explain clinical benefit observed with adenoidectomy
52Funding Support The Deafness Research Foundation Children’s Research Center of MichiganFMREDepartment of OtolaryngologyLions Hearing Center of Michigan
53Research 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.