The continuum of biofilm-like structures in different ...chronic suppurative otitis media (CSOM),...

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Poster Design & Printing by Genigraphics ® - 800.790.4001 Dai Shibata 1,2 , Patricia A. Schachern 1 , Vladimir Tsuprun 1 , Tamotsu Harada 2 , Michael M. Paparella 3 , Sebahattin Cureoglu 1 Department of Otolaryngology, University of Minnesota, Minneapolis, MN, 1 Department of Otolaryngology, Kawasaki Medical School, Kurashiki, Japan, 2 Paparella Ear Head and Neck Institute, Minneapolis, MN 3 DISCUSSION ABSTRACT Subjects : 38 temporal bones (TBs) with SOM, 23 with MOM, 30 with POM, and 30 with COM with purulent effusion were selected. TBs studied are from the collection at the University of Minnesota (Minneapolis, MN, USA). All of the TBs were removed at autopsy, fixed in 10% buffered formalin solution, decalcified, and embedded in celloidin. Each TB was serially sectioned in the horizontal plane at a thickness of 20 μm. Every 10th section was stained with hematoxylin and eosin and mounted on a glass slide for light microscopic study. Additional sections were stained with Weigert-Gram stain to ascertain the presence of bacteria in the TB. Classification of otitis media was based on properties of fluid and pathological changes (Table 1). Furthermore we classified SOM and MOM each into two types by whether they have a purulent portion. All COM TBs which we selected in this study have purulent middle ear fluid. Cases with history of immunosuppressive drugs, otological surgery, leukemia, or other otological or systemic diseases were excluded. ACKNOWLEDGEMENTS CONCLUSION RESULTS MATERIALS & METHODS A biofilm is an aggregate of microorganisms, in which cells adhere to each other on a surface with an extrapolysaccharide matrix. The biofilm matrix provides protection from environmental threats including antibiotics, surfactants, and host immune responses. Biofilms are believed to play a role in chronic diseases and have been associated with chronic otitis media (COM). Histopathological studies of otitis media (OM) have classified the properties of middle ear effusions as serous OM (SOM), mucoid OM (MOM), and purulent OM (POM). It is recognized that the various types of OM follow a continuum with early forms of the disease leading to more chronic forms over time (Paparella 1984. In this study, we examine the occurrence, frequency, and location of biofilm-like structures in the middle ear and mastoid of human temporal bones with different types of OM to investigate if these structures play a role in the continuum of pathological changes in the progression of OM. Objectives: It is recognized that the various types of otitis media (OM) follow a continuum with early forms of the disease leading to more chronic forms over time. This study investigates if biofilms play a role in the continuum of pathological changes and the progression of OM. Methods: 38 human temporal bones (TBs) with SOM, 23 MOM, 30 POM, and 30 COM with purulent effusion, were selected. We defined “biofilm-like structures” as bacteria and their aggregates bound to fibrous material in “web-like structures” that contain inflammatory cells. We examined the frequency and anatomical locations of biofilm-like structures. Results: Biofilm-like structures were observed in 9 (23.7%) of the 38 SOM, 10 (43.5%) of the 23 MOM, 17 (56.7%) of 30 POM, and 20 (66.7%) of 30 COM. There was a significant difference between SOM and POM, and SOM and COM. The most common anatomical location was mastoid antrum and cells. The number of anatomical locations with biofilm-like structures in each ear was 2.11 ± 0.93 in SOM, 2.50 ± 1.51 in MOM, 2.71 ± 1.57 in POM and 4.15 ± 1.60 in COM. A significant difference between COM and SOM, COM and MOM, and COM and POM, was observed. Conclusion: Given the similar incidence in the clinical studies and in our histopathologic findings, biofilm-like structures observed in our celloidin-embedded human TBs may indeed be biofilms. The growth and development of biofilms may play an important role in the progression of chronicity of OM. INTRODUCTION The continuum of biofilm-like structures in different types of otitis media This study was supported by the NIDCD 3U24 DC008559-03S109, R01 DC006452, U24 DC011968-01, 5M Lions International, the International Hearing Foundation, and the Starkey Foundation. Table 2 No. of TBs Side Rt / Lt No. of TBs with biofilm-like structures SOM 38 21 / 17 9 (23.7%) pure SOM 8 4 / 4 0 (0.0%) sero-purulent OM 30 17 / 13 9 (30.0%) MOM 23 10 / 13 10 (43.5%) pure MOM 7 4 / 3 2 (28.6%) muco-purulent OM 16 6 / 10 8 (50.0%) POM 30 15 / 15 17 (56.7%) COM 30 13 / 17 20 (66.7%) P < 0.01 P < 0.001 1. The presence and frequency of biofilm-like structures. The results of the frequency of biofilm-like structures in each type of OM are shown in Table 2. We found biofilm-like structures in all types of OM (Fig. 1). A significant difference in the frequency of biofilm-like structures between SOM and POM (p = 0.0055), and SOM and COM (p < 0.001), was observed by chi-square test. SOM and MOM were each classified into two groups those with a purulent component to the effusion and those without. In SOM cases, biofilm-like structures were shown in only SPOM; we did not find them in pure SOM. In MOM cases, we found biofilm-like structures in both types of MOM. Figure 1 Biofilm-like structures in OM. (A) and (B) show biofilm-like structures around the oval window in a case with POM. Area boxed in (A) is enlarged in (B). (C) and (D) show biofilm-like structures in mastoid cells with COM. Area boxed in (C) is enlarged in (D). Bacterial bodies (black arrowheads), a network of fibrous material, and host inflammatory cells (black arrows) are observed. Co indicates cochlea; FN, facial nerve; TM, tympanic membrane; V, vestibule. (E) and (F) show biofilm-like structures stained with Weigert-Gram stain in COM case. Area boxed in (E) is enlarged in (F). Single bacterial bodies (black arrowheads) and bacterial clusters (white arrows) are observed in biofilm-like structures. (A) - (D) were stained with Hematoxylin and eosin. (E) and (F) were stained with Weigert-Gram stain. 3. Number of anatomical locations with biofilm-like structures in each TB. We counted the number of anatomical locations with biofilm-like structures in each TB, and compared this number in each type of OM (Table 3). There was a significant difference in the number of locations by student t test between COM and SOM (p = 0.0015), COM and MOM (p = 0.011), and COM and POM (p = 0.0091). 2. Location and number of TBs with biofilm-like structures in each type of OM. We counted the number of TBs with different types of OM in each anatomical location (Fig. 2). The most common anatomical location was mastoid antrum and cells (36 of 56 TBs, 64.3%). The rarest anatomical location was the Eustachian tube (2 of 56 TBs, 3.6%). Figure 2 Number of TBs with biofilm-like structures in each anatomical location. Given the similar incidence in the clinical studies and in our histopatholgic findings, biofilm-like structures observed in our celloidin-embedded human TBs may indeed be biofilms. The growth and development of biofilms may play an important role in the progression of the chronicity of OM. A biofilm is a structured community of microorganisms embedded in an extra- polysaccharide matrix. The biofilm matrix enhances the ability of microbes to evade host defenses and antibiotic medication. The recent elucidation of biofilms discloses that biofilms play an important role in otorhinolaryngological diseases. Previous studies of biofilms in human middle ears used small mucosal specimens removed during surgical biopsy. To our knowledge, there are no previous reports about the frequency and location of biofilms within the whole of the human middle ear cleft. Biofilms reported in surgical biopsies and in chinchilla models of bacterial otitis media describe them histopathologically as bacteriial aggregates embedded within a fibrous matrix (Reid et al 2009, Byrd et al 2011). Because our TBs were fixed and embedded, it is not possible to do some of the traditional methods to verify the presence of biofilms; however, our histological and histochemical examinations did confirm the presence of bacteria and their aggregates, fibrous matrix, and inflammatory cells, characteristic of the bacterial biofilms described in chinchillas. Furthermore, the percentage of COM cases with biofilms seen in our study is also similar to that reported for samples in human surgical biopsies. Lee et al. (2009) reported the frequency of biofilm was 60% in chronic suppurative otitis media (CSOM), and Lampikoski et al. (2012) also reported 66% in mastoid mucosa with CSOM. In our study, biofilm-like structures were found in 66.7% in COM. Given the similar incidence in the clinical studies and in our histopatholgic findings, it is reasonable to believe that these structures may indeed be biofilms. In our study, we observed biofilm-like structures in all types of OM, and in some anatomical locations where the presence of biofilms have not yet been reported. To our knowledge, the presence of biofilms in human middle ears was reported in small mucosal specimens around the promontory, mastoid tissues, and some abnormal tissues (e.g. cholesteatoma and granulation tissue). We found the most common anatomical location was the mastoid antrum and cells, and the least common was the Eustachian tube. Our findings suggest that biofilm-like structures are more easily generated in small and narrow spaces, and areas of poor aeration. Biofilm-like structures were not seen in any of our cases of pure SOM, while their highest incidence occurred in COM. Paparella et al. (1990) described OM as a continuum, where the more acute serous phase can progress over time into the more chronic stages. We suggest that the growth and development of biofilms may play an important role in the progression of chronicity of OM. Our findings of the highest incidence occurring in those cases with COM support these concepts. Biofilm-like structures in each type of OM : We defined “biofilm-like structures” as bacteria and their aggregates bound to fibrous material in “web-like structures” that contain inflammatory cells. The presence of biofilm-like structures was examined in the following anatomical locations: epitympanum, mesotympanum, hypotympanum, Eustachian tube, facial recess, sinus tympani, the areas around the oval window, round window, and mastoid antrum and cells. All data was compared among each type of OM. Data was analyzed by using the chi-square test and Student t test. A p-value of less than 0.05 was considered to be significant. Table 3 Number of anatomical locations with biofilm-like structures in TBs with each type of OM (mean ± SD). SOM 2.11 ± 0.93 MOM 2.50 ± 1.51 POM 2.71 ± 1.57 COM 4.15 ± 1.60 P < 0.01 P < 0.05 P < 0.01 Table 1 Properties of fluid and pathological change SOM serous effusion with little inflammatory cell infiltration pure SOM only serous effusion without purulent portion sero-purulent OM both serous and purulent portion MOM fluid that contained mucoid strands and cellular fragments pure MOM only mucoid effusion without purulent portion muco-purulent OM both mucoid and purulent portion POM fluid occupied by many inflammatory cells and cellular fragments COM inflammation of the middle ear cleft that included at least one of the following types of tissue pathology: fibrocystic structures; granulation tissue; cholesterol granuloma; cholesteatoma; bony changes; and tympanosclerosis All COM TBs have purulent middle ear fluid in this study. Number of ears with biofilm-like structures

Transcript of The continuum of biofilm-like structures in different ...chronic suppurative otitis media (CSOM),...

Poster Design & Printing by Genigraphics® - 800.790.4001

Dai Shibata1,2, Patricia A. Schachern1, Vladimir Tsuprun1, Tamotsu Harada2, Michael M. Paparella3, Sebahattin Cureoglu1

Department of Otolaryngology, University of Minnesota, Minneapolis, MN,1 Department of Otolaryngology, Kawasaki Medical School, Kurashiki, Japan,2

Paparella Ear Head and Neck Institute, Minneapolis, MN3

DISCUSSION

ABSTRACT

Subjects: 38 temporal bones (TBs) with SOM, 23 with MOM, 30 with POM, and 30 with COM with purulent effusion were selected. TBs studied are from the collection at the University of Minnesota (Minneapolis, MN, USA). All of the TBs were removed at autopsy, fixed in 10% buffered formalin solution, decalcified, and embedded in celloidin. Each TB was serially sectioned in the horizontal plane at a thickness of 20 µm. Every 10th section was stained with hematoxylin and eosin and mounted on a glass slide for light microscopic study. Additional sections were stained with Weigert-Gram stain to ascertain the presence of bacteria in the TB.

Classification of otitis media was based on properties of fluid and pathological changes (Table 1). Furthermore we classified SOM and MOM each into two types by whether they have a purulent portion. All COM TBs which we selected in this study have purulent middle ear fluid. Cases with history of immunosuppressive drugs, otological surgery, leukemia, or other otological or systemic diseases were excluded.

ACKNOWLEDGEMENTS

CONCLUSION

RESULTS

MATERIALS & METHODS

A biofilm is an aggregate of microorganisms, in which cells adhere to each other on a surface with an extrapolysaccharide matrix. The biofilm matrix provides protection from environmental threats including antibiotics, surfactants, and host immune responses. Biofilms are believed to play a role in chronic diseases and have been associated with chronic otitis media (COM).

Histopathological studies of otitis media (OM) have classified the properties of middle ear effusions as serous OM (SOM), mucoid OM (MOM), and purulent OM (POM). It is recognized that the various types of OM follow a continuum with early forms of the disease leading to more chronic forms over time (Paparella 1984.

In this study, we examine the occurrence, frequency, and location of biofilm-like structures in the middle ear and mastoid of human temporal bones with different types of OM to investigate if these structures play a role in the continuum of pathological changes in the progression of OM.

Objectives: It is recognized that the various types of otitis media (OM) follow a continuum with early forms of the disease leading to more chronic forms over time. This study investigates if biofilms play a role in the continuum of pathological changes and the progression of OM.

Methods: 38 human temporal bones (TBs) with SOM, 23 MOM, 30 POM, and 30 COM with purulent effusion, were selected. We defined “biofilm-like structures” as bacteria and their aggregates bound to fibrous material in “web-like structures” that contain inflammatory cells. We examined the frequency and anatomical locations of biofilm-like structures.

Results: Biofilm-like structures were observed in 9 (23.7%) of the 38 SOM, 10 (43.5%) of the 23 MOM, 17 (56.7%) of 30 POM, and 20 (66.7%) of 30 COM. There was a significant difference between SOM and POM, and SOM and COM. The most common anatomical location was mastoid antrum and cells. The number of anatomical locations with biofilm-like structures in each ear was 2.11 ± 0.93 in SOM, 2.50 ±1.51 in MOM, 2.71 ± 1.57 in POM and 4.15 ±1.60 in COM. A significant difference between COM and SOM, COM and MOM, and COM and POM, was observed.

Conclusion: Given the similar incidence in the clinical studies and in our histopathologic findings, biofilm-like structures observed in our celloidin-embedded human TBs may indeed be biofilms. The growth and development of biofilms may play an important role in the progression of chronicity of OM.

INTRODUCTION

The continuum of biofilm-like structures in different types of otitis media

This study was supported by the NIDCD 3U24 DC008559-03S109, R01 DC006452, U24 DC011968-01, 5M Lions International, the International Hearing Foundation, and the Starkey Foundation.

Table 2 No. of TBs

Side Rt / Lt

No. of TBs with biofilm-like structures

SOM 38 21 / 17 9 (23.7%)pure SOM 8 4 / 4 0 (0.0%)sero-purulent OM 30 17 / 13 9 (30.0%)

MOM 23 10 / 13 10 (43.5%)pure MOM 7 4 / 3 2 (28.6%)muco-purulent OM 16 6 / 10 8 (50.0%)

POM 30 15 / 15 17 (56.7%)COM 30 13 / 17 20 (66.7%)

P < 0.01P < 0.001

1. The presence and frequency of biofilm-like structures.The results of the frequency of biofilm-like structures in each type of OM are

shown in Table 2. We found biofilm-like structures in all types of OM (Fig. 1). A significant difference in the frequency of biofilm-like structures between SOM and POM (p = 0.0055), and SOM and COM (p < 0.001), was observed by chi-square test.SOM and MOM were each classified into two groups those with a purulent component to the effusion and those without. In SOM cases, biofilm-like structures were shown in only SPOM; we did not find them in pure SOM. In MOM cases, we found biofilm-like structures in both types of MOM.

Figure 1 Biofilm-like structures in OM. (A) and (B) show biofilm-like structures around the oval window in a case with POM. Area boxed in (A) is enlarged in (B). (C) and (D) show biofilm-like structures in mastoid cells with COM. Area boxed in (C) is enlarged in (D). Bacterial bodies (black arrowheads), a network of fibrous material, and host inflammatory cells (black arrows) are observed. Co indicates cochlea; FN, facial nerve; TM, tympanic membrane; V, vestibule. (E) and (F) show biofilm-like structures stained with Weigert-Gram stain in COM case. Area boxed in (E) is enlarged in (F). Single bacterial bodies (black arrowheads) and bacterial clusters (white arrows) are observed in biofilm-like structures. (A) - (D) were stained with Hematoxylin and eosin. (E) and (F) were stained with Weigert-Gram stain.

3. Number of anatomical locations with biofilm-like structures in each TB.

We counted the number of anatomical locations with biofilm-like structures in each TB, and compared this number in each type of OM (Table 3). There was a significant difference in the number of locations by student t test between COM and SOM (p = 0.0015), COM and MOM (p = 0.011), and COM and POM (p = 0.0091).

2. Location and number of TBs with biofilm-like structures in each type of OM.

We counted the number of TBs with different types of OM in each anatomical location (Fig. 2). The most common anatomical location was mastoid antrum and cells (36 of 56 TBs, 64.3%). The rarest anatomical location was the Eustachian tube (2 of 56 TBs, 3.6%).

Figure 2 Number of TBs with biofilm-like structures in each anatomical location.

Given the similar incidence in the clinical studies and in our histopatholgic findings, biofilm-like structures observed in our celloidin-embedded human TBs may indeed be biofilms. The growth and development of biofilms may play an important role in the progression of the chronicity of OM.

A biofilm is a structured community of microorganisms embedded in an extra-polysaccharide matrix. The biofilm matrix enhances the ability of microbes to evade host defenses and antibiotic medication. The recent elucidation of biofilms discloses that biofilms play an important role in otorhinolaryngological diseases.

Previous studies of biofilms in human middle ears used small mucosal specimens removed during surgical biopsy. To our knowledge, there are no previous reports about the frequency and location of biofilms within the whole of the human middle ear cleft. Biofilms reported in surgical biopsies and in chinchilla models of bacterial otitis media describe them histopathologically as bacteriial aggregates embedded within a fibrous matrix (Reid et al 2009, Byrd et al 2011). Because our TBs were fixed and embedded, it is not possible to do some of the traditional methods to verify the presence of biofilms; however, our histological and histochemical examinations did confirm the presence of bacteria and their aggregates, fibrous matrix, and inflammatory cells, characteristic of the bacterial biofilms described in chinchillas. Furthermore, the percentage of COM cases with biofilms seen in our study is also similar to that reported for samples in human surgical biopsies. Lee et al. (2009) reported the frequency of biofilm was 60% in chronic suppurative otitis media (CSOM), and Lampikoski et al. (2012) also reported 66% in mastoid mucosa with CSOM. In our study, biofilm-like structures were found in 66.7% in COM. Given the similar incidence in the clinical studies and in our histopatholgic findings, it is reasonable to believe that these structures may indeed be biofilms.

In our study, we observed biofilm-like structures in all types of OM, and in some anatomical locations where the presence of biofilms have not yet been reported. To our knowledge, the presence of biofilms in human middle ears was reported in small mucosal specimens around the promontory, mastoid tissues, and some abnormal tissues (e.g. cholesteatoma and granulation tissue). We found the most common anatomical location was the mastoid antrum and cells, and the least common was the Eustachian tube. Our findings suggest that biofilm-like structures are more easily generated in small and narrow spaces, and areas of poor aeration.

Biofilm-like structures were not seen in any of our cases of pure SOM, while their highest incidence occurred in COM. Paparella et al. (1990) described OM as a continuum, where the more acute serous phase can progress over time into the more chronic stages. We suggest that the growth and development of biofilms may play an important role in the progression of chronicity of OM. Our findings of the highest incidence occurring in those cases with COM support these concepts.

Biofilm-like structures in each type of OM: We defined “biofilm-like structures” as bacteria and their aggregates bound to fibrous material in “web-like structures” that contain inflammatory cells. The presence of biofilm-like structures was examined in the following anatomical locations: epitympanum,mesotympanum, hypotympanum, Eustachian tube, facial recess, sinus tympani, the areas around the oval window, round window, and mastoid antrum and cells.

All data was compared among each type of OM. Data was analyzed by using the chi-square test and Student t test. A p-value of less than 0.05 was considered to be significant.

Table 3 Number of anatomical locations with biofilm-like structures in TBs with each type of OM (mean ± SD).

SOM 2.11 ± 0.93

MOM 2.50 ± 1.51

POM 2.71 ± 1.57

COM 4.15 ± 1.60P < 0.01

P < 0.05P < 0.01

Table 1 Properties of fluid and pathological change

SOM serous effusion with little inflammatory cell infiltration

pure SOM only serous effusion without purulent portion

sero-purulent OM both serous and purulent portion

MOM fluid that contained mucoid strands and cellular fragments

pure MOM only mucoid effusion without purulent portion

muco-purulent OM both mucoid and purulent portion

POM fluid occupied by many inflammatory cells and cellular fragments

COM

inflammation of the middle ear cleft that included at least one of the following types of tissue pathology: fibrocystic structures; granulation tissue; cholesterol granuloma; cholesteatoma; bony changes; and tympanosclerosisAll COM TBs have purulent middle ear fluid in this study.

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