Location of Bacterial Biofilm in the Mucus Overlying the Adenoid by Light Microscopy

ORIGINAL ARTICLE Location of Bacterial Biofilm in the Mucus Overlying the Adenoid by Light Microscopy

Birgit Winther, MD; Brian C. Gross, MD; J. Owen Hendley, MD; Stephen V. Early, MD

Objective: To determine the location of bacteria and tion protocol. One adenoid that was missing the surface biofilm in adenoid tissue and in mucus overlying the epithelium was excluded from further evaluation. adenoid. Main Outcome Measure: Identification of bacteria by light microscopy. Design: Adenoids removed in 1 piece were oriented to the cephalic and caudal ends. Mucus was fixed by the Results: Bacteria in large numbers were present in the gradual addition of Carnoy fluid. Consecutive histo- mucus overlying the surface of all 9 adenoids; bacteria were logic sections were stained with periodic acid–Schiff for not found in the parenchyma of the adenoids below the visualization of the exopolysaccharide matrix, Giemsa for epithelial surface. Bacterial biofilms were present on 8 of visualization of bacteria and cells, and fluorescent in situ the 9 adenoids. Sessile (attached) biofilm was present on hybridization with a universal probe for visualization of the caudal end of only 1 adenoid. Multiple planktonic (un- bacteria. attached) biofilms were present on 7 adenoids, always in areas not subject to mucus flow. Biofilms were most com- Setting: Department of Otolaryngology–Head and Neck mon on the caudal portions of adenoids. Surgery, University of Virginia. Conclusions: Bacteria of the adenoid reside in secretions on the surface and in crypts. Biofilms, predomi- Participants: We obtained adenoids from children 10 nantly planktonic, were present on 8 of 9 adenoids ex- years or younger who had chronic adenotonsillitis or ob- cised because of hypertrophy. Whether biofilms have a structive sleep apnea. Twenty-seven adenoids were used role in the causation of adenoid hypertrophy is not known. to develop the fixation method. We examined histologic sections from 9 of 10 adenoids fixed using the final fixa- Arch Otolaryngol Head Neck Surg. 2009;135(12):1239-1245

HE ADENOID HAS STRUC- face of adenoids; some bacteria were ture and function that are present in the secretion and on the sur- similar in many ways to face of the excised adenoids in histologic those of a lymph node.1 Mu- sections after formalin fixation. In the ini- cus from the nose flows tial phase of the present study, bacteria acrossT the surface of the adenoid, which were not found in formalin-fixed tissue in is in the midline on the posterior wall of the adenoid parenchyma or on the ad- the nasopharynx. Adenoid hypertrophy/ enoid surface with fluorescent in situ hy- hyperplasia, common in young children, bridization (FISH) using a universal bac- may lead to adenoidectomy. One justifi- terial probe.3 cation for removal is the concept that For the present study we hypoth- chronic infection of the adenoid may lead esized that the bacteria of the adenoid re- to hypertrophy, necessitating removal to side in the mucus overlying the adenoid, clear the infection. Aerobic bacteria in large which is often lost during routine fixa- quantities can be cultured easily from the tion. The approach to the hypothesis was surface of the adenoid, but bacterial in- facilitated by the work of Swidsinski et al,4 Author Affiliations: fection in the substance (parenchyma) of who used Carnoy fluid on biopsy samples Departments of the adenoid has not been demonstrated. of colon mucosa to demonstrate bacteria Otolaryngology–Head and Neck Ivarsson and Lundberg2 demonstrated the in the mucus overlying colonic epithe- Surgery (Drs Winther, Gross, and Early) and Pediatrics presence of many bacteria and polymor- lium. The use of Carnoy fluid, based on 5,6 (Dr Hendley), University of phonuclear leukocytes (PMNs) with in- previous work, was crucial in preserva- Virginia School of Medicine, gested bacteria in Giemsa-stained touch tion of bacteria in the mucus layer on mu- Charlottesville. preparations of secretions from the sur- cosal surfaces because the mucus layer was

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©2009 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/26/2021 means for identification of bacteria consisted of FISH with A a universal bacterial probe. Bacterial biofilms are defined as aggregates of bacteria “embedded in a matrix of extracellular polymeric substances.”7 The 2 types are sessile biofilm, which is adherent to a surface, and planktonic biofilm (sludge flocs), which is roughly spherical in shape and is not attached to a surface. The community of bacteria in biofilm is surrounded by an extracellular substance com- monly composed of polysaccharides, protein, and nucleic acid.8 For the purpose of locating bacteria and biofilm in this study, consecutive histologic sections from each paraffin block of adenoid tissue were stained with periodic acid–Schiff (PAS) for visualization of the exopolysaccharide matrix, Giemsa for visualization of bacteria and cells, and FISH for specific detection of bacteria.

METHODS

ADENOID SPECIMENS

B Thirty-seven adenoids removed in 1 piece (Figure 1A) were used. The indications for adenoid removal were chronic adenotonsillitis or obstructive sleep apnea. The use of the specimens was exempt from the need for informed consent because they were considered discarded material by the Institutional Review Board for Health Sciences Research at the University of Virginia; no identifying information on the patients was available other than age (Յ10 years). We used 27 adenoids to develop a protocol for the reten- tion of bacteria and mucus on the adenoid surface so that the relationship of bacteria to the adenoid surface could be as- sessed. The histologic findings on 9 of 10 adenoids fixed using C Nasopharyngeal lumen the final protocol are described in the “Results” section. One F F adenoid that was missing the surface epithelium was excluded F F from further evaluation. F C I C I V I C V V V DEVELOPMENT OF FIXATION PROTOCOL V Fixative M

Figure 1. Adenoid removed from a child 10 years or younger with chronic We used Carnoy fluid as a fixative throughout. Carnoy fluid adenotonsillitis or obstructive sleep apnea. A, Gross appearance of the adenoid contains ethanol, chloroform, and acetic acid. The ethanol con- removed in 1 piece and oriented to the cephalic/caudal ends. Lines indicate 3 centration is 60%,3-5 but the acetic acid concentration may be cross-section cuts (cephalic, middle, and caudal). B, Whole mount of a cross 30% with 10% chloroform6 or 10% with 30% chloroform.9 Three section, stained with periodic acid–Schiff (PAS). The adenoid has opened up adenoids were cut in half; half was fixed with the 10% acetic like a fan, revealing interfold spaces. C, Schematic of a cross section of adenoid. Each fold (F) consists of a vascular core (V), surrounded by lymphoid acid formulation and the other half with the 30% acetic acid follicles (not shown on the schematic; seen on the PAS-stained section in B). formulation. Fixation with the 30% acetic acid version re- Five folds are depicted. The epithelium on top of the folds forms the sulted in better preservation of the mucus on the surface. There- nasopharyngeal surface of the adenoid. The horizontal cut through the adenoid fore, Carnoy fluid with 60% ethanol, 30% acetic acid, and 10% after fixation allows the folds to separate from each other like a fan, revealing chloroform was adopted as the standard fixative; tissue was fixed the interfold space (I). This space is very narrow before the cross section is for 2 hours. cut. It is lined by adenoid epithelium and may contain cells in fluid or mucus. Adenoid crypts (C) are narrow epithelium-lined cul de sacs that may open onto nasopharyngeal surface or the interfold wall. Crypts may extend very deeply Application of Fixative into the fold tissue. The vascular core of each fold contains blood vessels and is surrounded by a “string of pearls” that consists of primary and secondary Adenoids placed in fixative in a bottle did not have mucus with lymphoid follicles covered by specialized adenoid epithelium.1 Mucus-secreting glands (M) (stained with PAS in B) are seen in the base of the folds. Glandular bacteria on the surface because the mucus had washed off be- ducts open onto the epithelial surface at the base of the interfold spaces. fore fixation. Subsequently, adenoids were placed with the cut (posterior) surface on filter paper in a Petri dish; fixative was added to the dish so that the mucus on the nasopharyngeal sur- 4 lost during formalin fixation. In the present work, we face might be fixed in place by acetic acid fumes before the level used Carnoy fluid to fix surface mucus in place to allow of fixative was high enough to cover the tissue. Adenoids fixed visualization of bacteria on the adenoid. The definitive in this way for 2 hours before being cut into smaller pieces for

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©2009 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/26/2021 embedding had flakes of fixed mucus, but the fixed mucus was RESULTS often dislodged during cutting. Sectioning the adenoids before fixation caused cells from the interior of the adenoid to be squeezed out onto the surface. ANATOMY OF THE ADENOID Finally, Carnoy fluid was added gradually during a 30- IN CROSS SECTION minute period to the adenoid on the filter paper. At 30 minutes, the surface with overlying mucus was fixed, but the in- Each adenoid specimen was removed as 1 piece and terior of the adenoid was not. The tissue was cut into smaller placed on filter paper with the cephalic/caudal orienta- pieces that were placed in cassettes without sponges before being returned to Carnoy fluid for the remaining 90 minutes re- tion (Figure 1A). After gradual fixation in Carnoy fluid quired for thorough fixation. The cassettes were then pro- for 30 minutes to retain the surface mucus, 3 horizontal cessed for embedding. cuts through the specimen yielded cephalic, middle, Adenoids examined with this technique demonstrated that and caudal sections. The horizontal cut through the flocs (planktonic biofilm) were present in the fixed mucus of adenoid folds allowed the adenoid to spread (open up) some adenoids, but the location on the adenoid and the num- like a fan, revealing interfold spaces lined by adenoid ber of areas involved were not apparent. This led to formula- epithelium that were hidden in the intact specimen tion of a final protocol in which the surgeon at the time of re- (Figure 1B and C). Adenoid epithelium on top of the moval (S.V.E.) placed the adenoid on the filter paper with the folds faces the nasopharyngeal lumen; most of the cephalic/caudal orientation noted on the paper. adenoid epithelium lines the interfold spaces. Each fold has a central core containing blood vessels. Between the FINAL STUDY PROTOCOL vascular core and the epithelium are primary and secondary lymphoid follicles with the germinal zone facing Ten adenoids oriented to the cephalic/caudal direction (Figure 1) were placed on filter paper (labeled cephalic/caudal) in a Petri specialized adenoid epithelium (Figure 1B). In the fold dish. Specimens were transported to the laboratory within 30 min- base are abundant mucoid-secreting glands containing utes; Carnoy fluid (with 30% acetic acid) was gradually added. PAS-positive material (Figure 1B). Narrow adenoid After 30 minutes of fixation, the adenoid was cut into 3 horizon- crypts open onto the nasopharyngeal surface and the tal cross sections (cephalic, middle, and caudal) that were placed interfold spaces. in cassettes for fixation for another 90 minutes. After fixation, cas- Mucus carried by mucociliary clearance from the na- settes were transferred to 70% ethanol and processed for paraf- sal cavity is deposited on the nasopharyngeal surface of fin embedding. Serial 4-µm sections were cut, and histologic sec- the adenoid. The mucus from the nose may enter and tra- tions from the cephalic, middle, and caudal portions of the adenoid verse the interfold spaces of the adenoid and exit from were each placed on a poly-L-lysine–coated slide. the caudal portion.

STAINING LOCATION OF BACTERIA ON ADENOID SPECIMENS Sections on slides were stained with hematoxylin-eosin (HE) and PAS using routine methods. The PAS stains proteogly- cans and polysaccharides; biofilm could be readily located by Three horizontal sections (cephalic, middle, and cau- the blue-purple color of the exopolysaccharide matrix. Bacte- dal) from 9 adenoids were studied for a total of 27 samples. ria were stained with Giemsa to allow visualization of cells in The surface epithelium of 1 adenoid was not intact, and addition to the bacteria. Brown and Hopps staining for bacte- it was omitted from further evaluation. To locate bacteria applied to some sections was not as useful as Giemsa be- ria, consecutive 4-µm histologic sections from each sample cause cells were difficult to identify. Fluorescent in situ hy- were stained with PAS, Giemsa, and FISH with the uni- bridization with a universal bacterial probe was used as the versal bacterial probe. Clusters of bacteria were easily vi- definitive method for identifying bacteria. sualized at a magnification of ϫ400 with FISH; single bacteria were sought by examination under oil immersion FISH FOR BACTERIA (magnification ϫ1000).

10 A previously described method for staining bacteria in paraf- Submucosa and Nasopharyngeal Surface fin-embedded tissue sections was followed exactly except for omission of the digestion of sections with proteinase K. The an- tisense oligonucleotide probe 5Ј-F-ACTGCTGCCTCCCGT- No bacteria, single or in clusters, were detected below AGGAGTTTATTCCTT-3Ј with 1 molecule of fluorescein the adenoid epithelium despite extensive search under isothiocyanate per probe (Life Technologies, Carlsbad, Califor- a magnification of ϫ1000. Single and/or small clusters nia) is generic for bacterial 16S ribosomal RNA.10 The histologic of bacteria were found on the epithelial surface facing sections were dewaxed, rehydrated, air dried, and then hybrid- the nasopharyngeal lumen of all 9 adenoids. Rows of ized overnight at 37°C with the probe (10 ng/µL) in hybridiza- bacteria embedded in a thin film of mucus on the epi- tion buffer.10 Sections were mounted in a commercially available Ј thelial surface of the top of folds were common medium with 4 ,6-diamidino-2-phenylindole (DAPI) (Vectash- (Figure 2). In addition, many bacteria were found ield; Vector Laboratories, Burlingame, California) and viewed dispersed in mucus on indentations of the nasopha- with UV light with a triple bandpass filter to identify blue staining (DAPI) of the cell nuclei and fluorescent green bacteria. ryngeal surface overlying the mouths of crypts and at Representative images were captured with the 3 color channels the openings of interfold spaces onto the nasopharyn- (red, green, and blue) by using a digital camera, and a composite geal surface. Few, if any, single bacteria were present was produced. Identification of single bacteria required viewing in the deeper half (farthest from the nasopharyngeal under oil immersion at a magnification of ϫ1000. lumen) of the interfold spaces.

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Figure 2. The thin layer of mucus covering the adenoid epithelium on the nasopharyngeal surface of a fold. A, With Giemsa staining, cells and bacteria Figure 3. Sessile biofilm is attached to the nasopharyngeal surface of a fold are shown embedded in the mucus layer (original magnification ϫ400). (adenoid 33). A, Periodic acid–Schiff stain shows the exopolysaccharide B, In the contiguous section, bacteria are stained with universal bacterial matrix. B, Fluorescent in situ hybridization with fluorescent bacterial probe of ϫ fluorescent in situ hybridization probe (original magnification ϫ1000). contiguous section stains bacteria in the matrix (original magnification 400).

Table. Biofilma on Cross Sections of the 8 Adenoids With A B Positive Staining Results

Cross Section Adenoid No. Cephalic Middle Caudal Biofilm Type 31 ϩϩϩSmall flocs 36 ϩ 0 ϩ Small flocs 37 ϩ 0 ϩ Large and small flocs 34 0 ϩ 0 Large flocs 28 0 0 ϩ Large and small flocs Figure 4. Planktonic biofilms (flocs) stained with periodic acid–Schiff. 29 0 0 ϩ Large flocs A, Large floc in the central interfold space of adenoid 29 (original 30 0 0 ϩ Small flocs magnification ϫ50). B, Several small flocs in the entryway to the crypt ϫ 33 0 0 ϩ 2 Areas of sessile biofilm in opening into an interfold space of adenoid 31 (original magnification 200). shallow indentations on nasopharyngeal surface Total 3 2 7 exopolysaccharide matrix with embedded bacteria in an indentation at the opening of a crypt (Figure 3). Abbreviations: flocs, planktonic biofilm; ϩ, positive staining results. This sessile biofilm was present at the same site on the a Biofilm identified with a combination of periodic acid–Schiff and Giemsa surface through multiple histologic sections, confirm- stains and fluorescent in situ hybridization with a universal bacterial probe. ing that it was attached. Flocs (planktonic biofilm) were not attached to the ad- With Giemsa staining, PMNs with ingested bacteria enoid surface. Flocs were present in interfold spaces were frequently found in mucus at the mouths of inter- (Figure 4A) and in large cryptlike passages opening into fold spaces and on the nasopharyngeal surface of folds. the interfold spaces (Figure 4B); flocs were not found on However, the ingested bacteria did not stain with the FISH the nasopharyngeal surface where they would be washed probe for ribosomal RNA of bacteria, suggesting that they away by mucus flow. Large flocs (Figure 4A) were differ- were no longer metabolically active. ent in appearance from small flocs (Figure 4B). A representative large floc (Figure 5) was spherical. The cen- Biofilm tral portion appeared to consist of a matrix with few bacteria. The density of bacteria increased toward the pe- Eight of the 9 adenoids were found to have biofilm on riphery in a somewhat radiating formation (Figure 5C and the adenoid surface, which was readily detected by D). The sphere was surrounded by a layer of PMNs that PAS staining of the exopolysaccharide matrix in which appeared to be unable to enter the matrix where the bac- the community of bacteria was located (Table). The teria were embedded (Figure 5A). Staining of bacteria by bacteria in biofilm stained with Giemsa and with FISH with the probe for ribosomal RNA was intense in the FISH. Biofilm was found on the caudal sections of 7 periphery of the sphere with diminished staining in the adenoids, on the middle sections of 2, and on the center (Figure 5E). Other large flocs had FISH-positive bac- cephalic sections of 3. Five adenoids had biofilm on 1 teria distributed throughout the matrix (Figure 5F). section, 2 had biofilm on 2 sections, and 1 had biofilm In contrast to large flocs, small flocs had exopolysac- on all 3 horizontal sections. charide matrix with embedded bacteria that did not ap- Sessile biofilm was present on only 1 adenoid; pear to be organized, and they usually were not spheri- planktonic biofilm (flocs) was present on the other 7. cal (Figure 6). Small flocs were frequently found close Sessile biofilm, attached to a surface to resist being together in interfold spaces or in crypts emptying into washed away by mucus flow, was found on the caudal interfold spaces (Figure 4B) and were sometimes found section of adenoid number 33 (Table). Two areas on in the mouth of interfold spaces close to the junction with the nasopharyngeal surface of that section had the the nasopharyngeal surface (not shown).

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D E F

100 µm

Figure 5. Representative large planktonic biofilm. An omega-shaped fixation artifact is seen in all sections of adenoid 37. A and B, Periodic acid–Schiff stainof adenoid 37 (original magnification ϫ20 and ϫ400, respectively). C and D, Giemsa stain of adenoid 37 (original magnification ϫ200 and ϫ400, respectively). E, Fluorescent in situ hybridization (FISH) stain for bacteria in adenoid 37 (original magnification ϫ400). F, FISH stain of a floc from adenoid 31 (original magnification ϫ400).

COMMENT A B

This study has demonstrated 2 major findings about the relationship of bacteria to the adenoid using Car- noy fluid for the fixation of overlying mucus and FISH staining of bacteria. First, bacteria were not found in the adenoid parenchyma despite extensive search under a magnification of ϫ1000. Bacteria that had Figure 6. Two small planktonic biofilms in a narrow space between folds. been ingested by phagocytes (PMNs and macro- A, Periodic acid–Schiff stain demonstrates the exopolysaccharide matrix of phages) could have been inside cells below the the biofilm. B, Fluorescent in situ hybridization probe demonstrates bacteria in the biofilm. adenoid surface but were not detected because they were metabolically inactive as they were being killed. Second, the bacteria of the adenoid were located in the This study provides a context for viewing the findings overlying surface secretion, which confirms our reported by other investigators who have examined the hypothesis that preserving mucus during the fixation relationship of bacteria to the adenoid.2,11-14 In 1994, process is required for detection of the bacterial bio- Forsgren and coworkers11 used FISH and transmission elec- film by light microscopy. Staining of consecutive his- tron microscopy of frozen sections to demonstrate tologic sections with PAS, Giemsa, and FISH allowed Hemophilus influenzae in macrophagelike cells that they definitive localization of the bacteria in relation to the found in the subepithelial layers of 10 adenoids. They adenoid epithelium. All 9 adenoids had bacteria in speculated that the intracellular bacteria had entered by mucus on the epithelium on the top of folds facing the way of the specialized epithelium of crypts, had been in- lumen of the nasopharynx; all but 1 also had bacteria gested by macrophages where they resided, and might con- in biofilm. The location of the 2 types of biofilm dif- stitute a “cellular reservoir.” We did not detect bacteria fered: sessile biofilm was attached to the nasopharyn- in phagocytes in the subepithelial tissue associated with geal surface on top of a fold, whereas the more com- crypts, but our FISH probe for bacterial ribosomal RNA mon planktonic biofilm (flocs) was invariably in areas would be unlikely to stain metabolically inactive bacteria not subject to the mucus flow across the nasopharyn- that had been ingested by phagocytes. geal surface. Biofilms were more common on caudal Ivarrson and Lundberg2 used touch preparations to sections than on cephalic and middle sections, per- characterize cells and bacteria on the surface of ad- haps related to being at the terminus of mucociliary enoids from 24 children before excision; formalin-fixed clearance or to proximity of the caudal section to the histologic sections from the same adenoids were used to oropharynx. examine the adenoid parenchyma. They demonstrated

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©2009 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/26/2021 abundant bacteria, PMNs with ingested bacteria, and im- v PMNs pass through adenoid epithelium to the sur- munoglobulin-secreting cells in the secretion. Bacteria face, where they may ingest and kill bacteria. were not seen in the adenoid parenchyma in histologic v Surface bacteria can form biofilms in which they sections, but PMNs traversed the epithelial layer to reach may be protected from ingestion by PMNs and killing the surface. Our study confirms and extends the find- by antibiotics. ings of the Swedish study.2 v Sessile biofilms may form on the nasopharyngeal sur- Zuliani et al12 fixed adenoids from 16 patients (7 with face of the adenoid, where they are attached and resist chronic rhinosinusitis and 9 with obstructive sleep ap- removal by mucus flow. nea) with glutaraldehyde for examination with scan- v Planktonic biofilms, which are unattached, may form ning electron microscopy. They reported that an aver- in areas of stagnation, for example, areas with decreased age of 94.9% of the nasopharyngeal surface of adenoids mucus flow. from patients with chronic rhinosinusitis was covered with Phagocytes are important for preventing bacterial mature biofilms compared with 1.9% coverage of ad- penetration into the adenoid parenchyma, and they enoids from patients with obstructive sleep apnea. Ses- may limit bacterial growth on the adenoid surface. In sile biofilm (but not flocs) could have been visualized with the hypertrophied adenoids that are available for this technique. Our findings are not consistent with those 12 examination because they had been removed, biofilm of Zuliani et al because we found sessile biofilm at only formation is common and is predominantly in the 2 sites on the caudal section of 1 of the 9 adenoids form of flocs rather than sessile biofilm. It may be that examined. 13 biofilms are uncommon on normal adenoids, but Swidsinski et al fixed 50 adenoid and 70 tonsil adenoids from children without disease of the upper samples with Carnoy fluid and used FISH to character- respiratory tract were not available for comparison ize bacteria on the adenoid surface by making photo- with the hypertrophied adenoids that have been stud- graphs “consecutively from the entire tissue surface” un- ied. This limits our ability to conclude that the bio- ϫ der a magnification of 1000. They showed photographs films are instrumental in producing the hypertrophy. of FISH-stained planktonic biofilms, which they termed It appears that the adenoid is like the tonsil in that microabscesses. They also described bacteria extending into maintaining homeostasis in the tissue and overlying mu- adenoid and tonsil tissue in fissures and diffusely infil- cus is an ongoing battle among the bacteria, PMNs, and trating subepithelial tissue. Although each tissue sample macrophages.2,15 The effect of biofilm on the adenoid is was treated with another stain (PAS, HE, or Gram stain) not clear. in addition to FISH, the precise location of bacteria in fissures and “diffusely infiltrating” adenoid tissue in relation to the complexities of the adenoid surface was not Submitted for Publication: February 3, 2009; final re- illustrated. vision received April 21, 2009; accepted May 25, 2009. Most recently, Kania and coworkers14 studied Correspondence: Birgit Winther, MD, Department of Oto- 39 excised adenoids with Gram staining of formalin- laryngology–Head and Neck Surgery, University of Vir- fixed sections, scanning electron microscopy of ginia Health System, PO Box 800713, Charlottesville, VA glutaraldehyde-fixed tissue, and confocal laser scanning 22908 ([email protected]). microscopy of frozen sections fixed with acetone. With Author Contributions: Drs Winther, Gross, and Hend- Gram staining they found microcolonies of mostly cocci ley had full access to all the data in the study and take on the outer surface of the adenoid, particularly in responsibility for the integrity of the data and the accu- small depressions between normal-appearing cells. For racy of the data analysis. Study concept and design: Win- confocal laser scanning microscopy, bacteria were ther, Gross, and Hendley. Acquisition of data: Winther, stained with propidium iodide and glycocalyx was Gross, Hendley, and Early. Analysis and interpretation of stained with fluorescein-labeled concanavalin A to bind data: Winther, Gross, and Hendley. Drafting of the manu- to mannose residues. They reported that 21 of 39 script: Winther, Gross, and Hendley. Critical revision of adenoids (54%) showed evidence of biofilm and that the manuscript for important intellectual content: Win- bacteria and glycocalyx colocalized by confocal laser ther, Gross, Hendley, and Early. Statistical analysis: Hend- scanning microscopy. It is not clear that our findings ley. Obtained funding: Winther and Hendley. Administra- are comparable to theirs because of differences in fixative, technical, and material support: Winther, Hendley, tion technique and different staining specificity in the and Early. Study supervision: Winther and Hendley. confocal laser scanning microscopy work. Financial Disclosure: None reported. Funding/Support: This study was supported in part by Summation of the evidence from this and other stud- the Pendleton Pediatric Infectious Disease Laboratory at ies2,11,13,14 allows the following conclusions to be drawn the University of Virginia. about the relationship between the adenoid and bacteria. Previous Presentation: This study was presented in v Viable bacteria are not found in the adenoid paren- part at the 22nd Congress of the European Rhinologic chyma. Society and the 27th International Symposium of v Bacteria that penetrate the adenoid epithelium are Infection and Allergy of the Nose; June 17, 2008; ingested and killed by phagocytes, macrophages, and/or Crete, Greece. PMNs. Additional Contributions: Sheri VanHoose, MLT, em- v Bacteria in great profusion are present on the ad- bedded, cut, and stained the adenoid tissues per our speci- enoid surface, where they reside in secretions. fications. Ken Tung, MD, is the director of the Research

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©2009 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/26/2021 Histology Core Laboratory, University of Virginia, and of biofilms. In: Evans LV, ed. Biofilms: Recent Advances in Their Study and oversees the work of Ms VanHoose. Control. Amsterdam, the Netherlands: Harwood Academic Publishers; 2000: 19-34. 8. Wimpenny J. Structural determinants in biofilm formation. In: Evans LV, ed. Bio- films: Recent Advances in Their Study and Control. Amsterdam, the Nether- REFERENCES lands: Harwood Academic Publishers; 2000:35-50. 9. Clark G, ed. Staining Procedures Used by the Biological Stain Commission. 3rd 1. Winther B, Innes DJ. The human adenoid: a morphologic study. Arch Otolaryn- ed. Baltimore, MD: Williams & Wilkins Co; 1973. gol Head Neck Surg. 1994;120(2):144-149. 10. Steel JH, Malatos S, Kennea N, et al. Bacteria and inflammatory cells in fetal mem- 2. Ivarsson M, Lundberg C. Phagocytosis in the nasopharyngeal secretion by cells branes do not always cause preterm labor. Pediatr Res. 2005;57(3):404-411. from the adenoid. Acta Otolaryngol. 2001;121(4):517-522. 11. Forsgren J, Samuelson A, Ahlin A, Jonasson J, Rynnel-Dagöö B, Lindberg A. 3. Schlager TA, Hendley JO, Peters C. Absence of bacterial reservoirs in the blad- Haemophilus influenzae resides and multiplies intracellularly in human adenoid der epithelium of patients with chronic bacteriuria due to neurogenic bladder [pub- tissue as demonstrated by in situ hybridization and bacterial viability assay. In- lished online August 17, 2009]. J Urol. 2009;182(4)(suppl):1714-1719. doi:10 fect Immun. 1994;62(2):673-679. .1016/j.juro.2009.02.063. 12. Zuliani G, Carron M, Gurrola J, et al. Identification of adenoid biofilms in chronic 4. Swidsinski A, Weber J, Loening-Baucke V, Hale LP, Lochs H. Spatial organiza- rhinosinusitis. Int J Pediatr Otorhinolaryngol. 2006;70(9):1613-1617. tion and composition of the mucosal flora in patients with inflammatory bowel 13. Swidsinski A, Göktas Ö, Bessler C, et al. Spatial organization of microbiota in disease. J Clin Microbiol. 2005;43(7):3380-3389. quiescent adenoiditis and tonsillitis. J Clin Pathol. 2007;60(3):253-260. 5. Ota H, Katsuyama T. Alternating laminated array of two types of mucin in the 14. Kania RE, Lamers GEM, Vonk MJ, et al. Characterization of mucosal biofilms on human gastric surface mucous layer. Histochem J. 1992;24(2):86-92. human adenoid tissues. Laryngoscope. 2008;118(1):128-134. 6. Matsuo K, Ota H, Akamatsu T, Sugiyama A, Katsuyama T. Histochemistry of the 15. Ebenfelt A, Ericson LE, Lundberg C. Acute pharyngotonsillitis is an infection surface mucous gel layer of the human colon. Gut. 1997;40(6):782-789. restricted to the crypt and surface secretion. Acta Otolaryngol. 1998;118(2): 7. Flemming H-C, Wingender J, Griebe T, Mayer C. Physico-chemical properties 264-271.

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