10.14639/0392-100X-N0996
Objectıve. The aim of this study was to investigate the efficacy of N-acetylcysteine (NAC) on biofilm layers and on the course of disease in chronic otitis media. Methods. Twenty-five rats that were induced with chronic otitis media (COM) were separated into three groups. In Group 1 (N = 18), 0.2% ciprofloxacin + 0.1% dexamethasone sodium phosphate + 0.5 mg/ml NAC solution was locally injected to the right ear of the rats; in Group 2, (N=18) 0.2% ciprofloxacin + 0.1% dexamethasone sodium phosphate was locally injected to the left ear of the rats. No treatment was applied to either ear of rats in Group 3 (N = 5). Histopathological and scanning electron microscope (SEM) evaluations were performed in all groups. Results. SEM revealed biofilm formation in all COM induced groups. No significant difference was seen between groups 1 and 2 in terms of suppuration levels, fibrosis, inner ear involvement, infection staging and biofilm formation (p &gt; 0.05). Conclusıons. In this study, while histopathological and SEM evaluation revealed no effect of 0.5 mg/ml NAC on the biofilm layer in COM-induced rats, further studies with NAC at different concentrations are still needed on different types of experimental animals.
application/xml
1827-675X
996
6
en
Mehmet Hakan Korkmaz
https://creativecommons.org/licenses/by-nc-nd/4.0
ACTA Otorhinolaryngologica Italica
rat
The effect of N-acetyl cysteine on biofilm layers in an experimental model of chronic otitis media
Text.Serial.Journal
https://www.actaitalica.it/
40
Otology
2021-01-21
2020-07-17
2021-01-21
2021-01-21
1
Mehmet Hakan Korkmaz
2020
10.14639/0392-100X-N0996
Yıldırım Beyazıt University Ear Nose and Throat Department, Ankara, Turkey
1827-675X
6
rat
en
The effect of N-acetyl cysteine on biofilm layers in an experimental model of chronic otitis media
https://www.actaitalica.it/article/download/996/499
40
ACTA OTORHINOLARYNGOLOGICA ITALICA 2020;40:457-462; doi: 10.14639/0392-100X-N0996




Otology


The effect of N-acetyl cysteine on biofilm layers
in an experimental model of chronic otitis media
L’effetto della N-acetil cisteina sullo strato di biofilm in un modello sperimentale di otite
media cronica
Elif Ersoy Çallıoğlu1, Sami Berçin2, Gülçin Başdemir3, Muzaffer Kiriş2, İlkan Tatar4, Arzu Tuzuner5,
Tolga Oğuzhan6, Tuba Müderris7, Mustafa Fevzi Sargon8, Mehmet Hakan Korkmaz2
1
 Ankara Bilkent City Hospital Otolaryngology Department, Ankara, Turkey; 2 Yıldırım Beyazıt University Ear Nose and Throat
Department, Ankara, Turkey; 3 Okan University Pathology Clinics, İstanbul, Turkey; 4 Hacettepe University Anatomy Department,
Ankara, Turkey; 5 Başkent University Ear Nose and Throat Clinics, Ankara, Turkey; 6 Istanbul Medicalpark Hospital Ear Nose and
Throat Clinics, Istanbul, Turkey; 7 Izmir Atatürk Training and Investigation Hospital Microbiology Clinics, Izmir, Turkey; 8 Atılım University
Anatomy Department, Ankara, Turkey


SUMMARY
Objectıve. The aim of this study was to investigate the efficacy of N-acetylcysteine (NAC)
on biofilm layers and on the course of disease in chronic otitis media.
Methods. Twenty-five rats that were induced with chronic otitis media (COM) were sepa-
rated into three groups. In Group 1 (N = 18), 0.2% ciprofloxacin + 0.1% dexamethasone
sodium phosphate + 0.5 mg/ml NAC solution was locally injected to the right ear of the
rats; in Group 2, (N=18) 0.2% ciprofloxacin + 0.1% dexamethasone sodium phosphate was
locally injected to the left ear of the rats. No treatment was applied to either ear of rats in
                                                                                                    Received: July 17, 2020
Group 3 (N = 5). Histopathological and scanning electron microscope (SEM) evaluations
                                                                                                    Accepted: September 21, 2020
were performed in all groups.
Results. SEM revealed biofilm formation in all COM induced groups. No significant dif-              Correspondence
ference was seen between groups 1 and 2 in terms of suppuration levels, fibrosis, inner ear         Elif Ersoy Çallıoğlu
involvement, infection staging and biofilm formation (p > 0.05).                                    Ankara Bilkent City Hospital Otolaryngology
Conclusıons. In this study, while histopathological and SEM evaluation revealed no effect           Department, Ankara, Turkey
of 0.5 mg/ml NAC on the biofilm layer in COM-induced rats, further studies with NAC at              E-mail: elifersoy78@hotmail.com
different concentrations are still needed on different types of experimental animals.
                                                                                                    Funding
KEY WORDS: chronic otitis, biofilm, N-acetylcysteine, rat                                           The present study was supported financially by
                                                                                                    project bureau of Yıldırım Beyazıt University.
RIASSUNTO
                                                                                                    Conflict of interest
Obıettıvo. Lo scopo di questo studio è quello di valutare l’efficacia della N-acetilcisteina        The Authors declare no conflict of interest.
(NAC) sullo strato di biofilm e sul decorso della malattia nell’otite media cronica.
Metodo. Venticinque ratti con otite media cronica (COM) sono stati divisi in tre gruppi. Nel
gruppo 1 (N = 18) sono stati iniettati 0,2% di ciprofloxacina + 0,1% di desametasone sodio            How to cite this article: Ersoy Çallioğlu E, Ber-
                                                                                                      çin S, Başdemir G, et al. The effect of N-acetyl
fosfato + 0,5 mg / ml di soluzione NAC all’orecchio destro dei ratti; nel gruppo 2 (N = 18)
                                                                                                      cysteine on biofilm layers in an experimental
sono stati iniettati localmente 0,2% di ciprofloxacina + 0,1% di desametasone sodio fosfato           model of chronic otitis media. Acta Otorhi-
all’orecchio sinistro dei ratti. Nessun trattamento è stato applicato alle orecchie inoculate         nolaryngol Ital 2020;40:457-462. https://doi.
dei ratti nel gruppo 3 (N = 5, entrambe le orecchie). Sono state eseguite valutazioni istopa-         org/10.14639/0392-100X-N0996
tologiche e valutazioni al microscopio elettronico (SEM) mediante scansione.
Rısultatı. SEM ha documentato la formazione di biofilm in tutti i gruppi indotti con COM.           © Società Italiana di Otorinolaringoiatria
Nessuna differenza statisticamente significativa è stata osservata tra i gruppi 1 e 2 in termini    e Chirurgia Cervico-Facciale
di livelli di suppurazione, fibrosi, coinvolgimento dell’orecchio interno, stadiazione dell’infe-
                                                                                                                   OPEN ACCESS
zione e formazione di biofilm (p > 0,05).
Conclusıone. Anche se in questo studio la valutazione istopatologica e SEM non ha ri-               This is an open access article distributed in accordance with
velato alcun effetto del 0,5 mg/ml NAC sullo strato di biofilm nei ratti indotti dalla COM,         the CC-BY-NC-ND (Creative Commons Attribution-Non-
                                                                                                    Commercial-NoDerivatives 4.0 International) license. The
per arrivare a una conclusione migliore è necessario eseguire ulteriori studi con di NAC a          article can be used by giving appropriate credit and mentio-
diverse concentrazioni su differenti tipi di animali da esperimento.                                ning the license, but only for non-commercial purposes and
                                                                                                    only in the original version. For further information: https://
PAROLE CHIAVE: otite cronica, biofilm, N-acetilcisteina, ratto                                      creativecommons.org/licenses/by-nc-nd/4.0/deed.en




                                                                                                                                                            457
E. Ersoy Çallıoğlu et al.




Introduction                                                      in all rats under general anaesthesia using ketamine hydro-
                                                                  chloride (90 mg/kg) and xylazine hydrocloride (10 mg/kg).
Biofilm is a community of micrororganisms residing in a
                                                                  After otoscopic examination and otoacoustic emission
gelatinous layer with polymeric structures and its produc-
                                                                  tests were performed, 30 rats with normal findings were
tion starts by attaching to a surface 1. Biofilm formation
                                                                  included in the present study. As in previous studies with
may take place in lifeless surfaces in vivo or in vitro. The
                                                                  chronic otitis model 3,13,14, tympanic membranes of 25 rats
microorganisms in biofilms are more resistant to antimi-
                                                                  (TM) were perforated with a proportion of 75% and 106
crobial agents than planktonic cells, as they have barriers
                                                                  colony P. aeruginosa strains (strain - ATCC -27853) were
impeding contact with antimicrobial agents and decreasing
                                                                  inoculated into the middle ear via the perforation. The same
sensitivity 2. Biofilm has also the characteristic of protect-
                                                                  inoculation procedure was repeated one week later. Sub-
ing the organism from osmotic stress, phage debris, toxic
                                                                  sequently, microscopic examination was carried out twice
compounds and antibiotics.
                                                                  weekly for three weeks and rats were followed up without
Various bacterial proteins (biofilm) are produced by bacte-
                                                                  treatment. Three weeks after the last inoculation, rats were
ria in chronic otitis media (COM), which increase the adhe-
                                                                  examined under sterile conditions and external ear cultures
sion and penetration of them into middle ear mucosa 3. It
                                                                  were obtained. Otomicroscopic examination of all rats re-
is considered that in resistant COM, otorrhoea is possibly
                                                                  vealed COM and purulent discharge in the outer ear canal.
derived from biofilm layer 3. In cases of COM in which otor-
                                                                  The cultures demonstrated the presence of P. aeruginosa
rhoea and inflammation cannot be relieved in spite of medi-
                                                                  infection in all rats. Meanwhile, 2 rats were lost due to mal-
cal treatment, surgical intervention is used as an alternative
                                                                  nutrition. Treatments were then started; to right ears of 18
treatment which aim to destroy the damaged osteitic tissue.
                                                                  rats, (Group 1) 0.2% ciprofloxacin + 0.1% dexamethasone
N-acetyl cysteine (NAC) is a precursor of glutathione and
                                                                  sodium phosphate + 0.5 mg/ml NAC and to left ears 0.2%
produces neuroprotection by preventing oxidative dam-
                                                                  ciprofloxacin + 0.1% dexamethasone sodium phosphate
age 4,5. In addition to its primary use, it is also utilised in
                                                                  was administered (Group 2). These medical treatments
chronic bronchitis, cancer, paracetamol intoxication and
aspergilloma 6-8. NAC exerts an eradicating effect on bio-        were administered for 4 weeks twice a day locally by injec-
film layer produced by various bacteria 9-12. The effect of       tion in the external auditory canal. In Group 3, 10 ears of
NAC, on COM associated biofilm layer produced by pri-             5 rats were not administered any medical treatment after
mary pathogen P. aeruginosa, has been demonstrated 12. Al-        inoculation. In Group 4, 10 ears of 5 rats were investigated
though the effect of NAC on biofilm layer was demonstated         as a control group without undergoing any procedure. Ani-
in infections produced by P. aeruginosa in respiratory and        mals were decapitated after deep anaesthesia with the same
renal systems, its effect in COM treatment is controversial.      protocol.
The aim of the present study was to investigate the effect of
NAC on biofilm layer formation in COM produced in a rat           Histomorphological evaluation
model. We also investigated the role of NAC in preventing         After temporal bones were removed, they were fixed in
the involvement of the inner ear in COM.                          10% formalin and after decalcification paraffin blocks
                                                                  were prepared. 4 μm sections from paraffin blocks were
                                                                  stained with haematoxylin-eosin, PAS and masson tri-
Materials and methods                                             chrome stains. In the sections examined with light micro-
The present study was carried out with 30 healthy male            scope, lesions produced by infection in external ear, middle
Wistar albino rats at a weight of 180-220 gm with the ap-         ear, inner ear and the presence of mast cells and suppura-
proval of ethics committee of Ankara Training and Investi-        tion to determine improvement levels and development of
gation Hospital (13 May 2014, No. 0018). The principles of        fibrosis. As the variation in quality and quantity of lesions
Helsinki declaration for laboratory animals were applied.         made it difficult to obtain repeatable objective data, fibrosis
All animals were followed and cared in accordance with a          and suppuration, which are more suitable for quantitative
protocol approved by institutional animal care group that         evaluations, were evaluated semiquantitatively (-, +, ++,
was compliant with experimental ethical principles and            +++). Inner ear involvement was shown as present or ab-
animal protection laws in Turkey. Animals were isolated           sent (+, -).
in standard cages throughout the experimental period and          In addition, stage/level of infection was evaluated histomor-
were given food and water ad libitum.                             phologically; lesions produced by varying combinations of
                                                                  different histomorphological findings were classifed into
Surgical technique                                                acute suppurative (suppuration/fibrosis: ++ or +++/- or +)
The surgical procedure was carried out by the same surgeon        (Fig. 1) or chronic suppurative (suppuration/fibrosis: ++ or



458
                                                                                                         The effect of N-acetyl cysteine on biofilm layers




                                                                                  phosphate buffer (pH 7.4), fixed with 1% ozmium tetroxide
                                                                                  in phosphate buffer (pH 7.4) and dehydrated in increasing
                                                                                  alcohol concentrations. After dehydration, specimens were
                                                                                  dried and mounted on metal frames with double sided adhe-
                                                                                  sive tape. Subsequently, Bio-Rad (Hercules, CA) gold thick
                                                                                  layer 150-A ° k were sprayed to specimens. SEM images
                                                                                  were obtained with JEOL SEM ASID-10 (Tokyo, Japan)
                                                                                  electron miscrosope at 500X-3000X magnification range.
                                                                                  The presence of biofilm was determined using SEM mor-
                                                                                  phological findings such as three dimensional structure,
                                                                                  variability in the size of microorganisms embedded in pol-
                                                                                  ysaccaride matrix and residue of multiple layers of tissue
                                                                                  and microorganisms. 3 x 3 mm specimens sampled from
                                                                                  middle ear mucosa were examined. With this investigation,
                                                                                  the presence of biofilm was evaluated and in biofilm posi-
Figure 1. Group1 rats, acute suppurative inflammation in middle ear (ME);         tive specimens, similar to other studies in the literature 15,
there is no involvement in inner ear (İE), routine histological characteristics   and the presence of biofilm in less than 25% of all visual-
(suppuration +++, fibrosis-).                                                     ised surface areas was classified as (+) between 25-50% as
                                                                                  (++) and over 50% as (+++) SEM analysis.
+++/++ or +++) depending on the severity and proportion
                                                                                  Statistical evaluation
of suppuration and fibrosis.
                                                                                  Descriptive statistics were expressed with the number and
Ears in which suppuration/fibrosis were (+/+), (+/-) or
                                                                                  percentage of cases both for nominal and ordinal variables.
(-/+), were classified as healed/improved (Fig. 2), because
                                                                                  Nominal data were analysed with Fisher’s exact test, while
none of the ears undergoing treatment procedures were
                                                                                  ordinal data were analysed with Mann Whitney U test. Data
completely healed (suppuration/fibrosis: -/-).                                    analysis was performed using IBM SPSS version 17.0 soft-
                                                                                  ware (IBM Corporation, Armonk, NY, USA). A p < 0.05
Scanning electron microscopic (SEM) evaluation                                    was considered significant for all results.
Fresh specimens obtained from 5 ears of each group were
fixed in 2.5% glutaraldehyde for 24 hours, irrigated in
                                                                                  Results
                                                                                  In all rats with experimental COM, cultures obtained three
                                                                                  weeks after the last inoculation revealed the P. aeruginosa
                                                                                  infection.

                                                                                  Histomorphological findings
                                                                                  Except for the control group, which did not undergo any
                                                                                  procedure, in all ears and temporal bones varying degrees
                                                                                  of supurative or chronic inflammation, granulation tissue,
                                                                                  vascularisation, fibrous tissue development and partial
                                                                                  appearance of cholesteatoma were detected. Mast cell in-
                                                                                  volvement was observed in eustachian tubes and inner ears
                                                                                  and for the most part ossicles and tympanic membrane
                                                                                  were completely destroyed and were surrounded by suppu-
                                                                                  ration and fibrosis, accompanied by inner ear involvement.
                                                                                  Inner ear structures; cranial nerve VIII, spinal ganglions,
                                                                                  cochlear and vestibulare bones were partially or complete-
Figure 2. Group 1, residual necrotic lamellous bone (LB) fragments due to         ly destroyed and replaced b fibrous repair tissue filling the
destruction in middle ear (ME) bone wall, caused by infection at the recovery
                                                                                  vestibulare and tympanic ducts.
process. Overlying new woven bone (WB) tissue and thin fibrous wall are seen
on the surface of degenerated epithelium. Suppuration continues in the lümen      It was observed that the inner ear was completely destroyed
(suppuration +, fibrosis +).                                                      in a few ears in which suppuration disappeared and fibrosis



                                                                                                                                                    459
E. Ersoy Çallıoğlu et al.




Table I. Histopathological findings of cases undergoing only treatment or NAC plus treatment.
                                                                 Group 2                                Group 1                       p-value
    Suppuration                                                                                                                       0.963†
    -                                                           4 (22.2%)                              2 (11.1%)
    +                                                           3 (16.7%)                              8 (44.4%)
    ++                                                          4 (22.2%)                              0 (0.0%)
    +++                                                         7 (38.9%)                              8 (44.4%)
    Fibrosis                                                                                                                          0.963†
    -                                                           4 (22.2%)                               2 (11.1%)
    +                                                           5 (27.8%)                               4 (22.2%)
    ++                                                          3 (16.7%)                              11 (61.1%)
    +++                                                         6 (33.3%)                               1 (5.6%)
    Inner ear chacaracteristics                                                                                                       0.338‡
    Normal                                                      14 (77.8%)                             17 (94.4%)
    Involved                                                    4 (22.2%)                               1 (5.6%)
    Infections stage                                                                                                                  0.462†
    Improved                                                    5 (27.8%)                              10 (55.6%)
    Acute suppurative                                           7 (38.9%)                               1 (5.6%)
    Chronic suppurative                                         6 (33.3%)                               7 (38.9%)
†
    Mann Whitney U test; ‡ Fisher’s Exact test.



was very mild. It was also found that inner ear involvement                      Table II. SEM findings in groups 1 and groups 2.
was more marked in groups that did not undergo treatment                                               Group 2             Group 1             p-value
(Group 3) compared to the groups that underwent treatment                            SEM                                                        0.690†
(Group 1, Group 2) (Fig. 1). In the evaluation of osteoblas-                         +                      1 (20.0%)     2 (40.0%)
tic and osteoclastic activities, no difference was found be-                         ++                     4 (80.0%)     3 (60.0%)
tween Groups 1 and 2 (Fig. 2).                                                       +++                    0 (0.0%)      0 (0.0%)
There was no significant difference between NAC+treatment                        †
                                                                                     Mann Whitney U test.
group and only treatment group with respect to severity of
suppuration and fibrosis, inner ear involvement or stage of
infection (p > 0.05) (Tab. I). In group 3, no statistical com-                   In SEM findings of Group 1 rats, widespread biofilm was
parison was performed with groups 1 and 2 since exten-                           observed along with polymorphonuclear leukocytes.
sive suppuration, fibrosis, and inner ear involvement was
detected in all ears.
In the control group, which did not undergo any procedure,
suppuration, fibrosis and inner ear involvement were not
observed.

SEM findings
In SEM imaging, biofilm formation was established in
all groups. There was no significant difference between
Groups 1 and 2 in terms of biofilm formation (p > 0.05)
(Tab. II).
In the evaluation of SEM findings in Group 3, widespread
biofilm areas were observed on the middle ear mucosa. As
there was no treatment, together with polymorphonuclear
leukocytes, many free bacteria, bacteria debris and ECM
appearance were also observed (Fig. 3).                                          Figure 3. Group 1 rats, SEM appearance of biofilm layer.



460
                                                                                         The effect of N-acetyl cysteine on biofilm layers




                                                                  on primary pathogen of COM, i.e. P. aeruginosa, has been
                                                                  demonstrated in many in vivo and in vitro studies 12,16,17. For
                                                                  this purpose, the efficacy of NAC has been investigated at
                                                                  different concentrations. In addition to other routes, NAC
                                                                  may also be administered locally. In the study by Özcan et
                                                                  al. 18, in which the effect of NAC in myringosclerosis in rats
                                                                  was investigated, 0.6 mg and 1.2 mg topical NAC was used,
                                                                  and it was demonstrated that it prevents the development of
                                                                  myringosclerosis. In the study by El-Feky et al. 16, aiming
                                                                  to determine the effect of the addition of NAC to cipro-
                                                                  floxacin treatment for biofilms on the surface of uretheral
                                                                  stents, 2-4 mg/ml NAC was used, and it was determined
                                                                  that the development of mature biofilms declined. Zhao et
                                                                  al. 12 added NAC at 0.5 mg /ml and 1 mg /ml to ciprofloxa-
Figure 4. SEM appearance of biofilm layer in Group 2 rats.        cin treatment and found that NAC decreased biofilm layer
                                                                  produced by P. aeruginosa and production of extrapolysac-
                                                                  charides. However, in that study, its effect on P. aerugi-
                                                                  nosa was investigated in patients with respiratory tract in-
                                                                  fections. Similar to this study, we used NAC at 0.5 mg/ml
                                                                  for eradication of biofilms. Herein, we found no significant
                                                                  difference between groups 1 and groups 2 with regard to se-
                                                                  verity of suppuration and fibrosis, involvement of inner ear,
                                                                  stage of infection or SEM findings (p > 0.05) (Figs. 1, 2). It
                                                                  may be thought that this is due to differences in experimen-
                                                                  tal animals used, variations in dose, the infectious process
                                                                  in which NAC was used and different organs. In previous
                                                                  studies, NAC was used in vivo and in vitro in pseudomonal
                                                                  infections, and not in treatment of COM, which may also
                                                                  explain the discrepant results.
                                                                  It has been demonstrated in various studies that NAC en-
                                                                  hances osteoblastic activity and bone regeneration 19. How-
Figure 5. Control group SEM findings.
                                                                  ever, in the present study, in COM model, no difference
                                                                  was found between Group 1 and 2 in terms of osteoblastic
SEM findings were similar in Group 2 to those in Group            and osteoclastic activity (Fig. 2).
1. Widespread biofilm formation was observed along with           There are few studies in the literature on the effect of NAC
blood components (Fig. 4).                                        on COM treatment. In the only study carried out by Choe
In control group without chronic otitis (Group 4), the nor-       et al. 20, it was established that NAC with ciprofloxacin was
mal middle ear mucosa was observed without any biofilm            effective in the treatment of refractory COM. Nevertheless,
formation (Fig. 5).                                               their study was performed with a small number of patients
                                                                  with no histopathological and SEM evaluation, and their
                                                                  conclusion was based upon solely clinical evaluation.
Discussion                                                        Severe and destructive lesions developing in all of the ears
To our knowledge, the present study is the first attempting       undergoing experimental treatment procedures made it dif-
to experimentally demonstrate the effect of NAC on bio-           ficult to evaluate the severity of histomorphological findings
film formation in the middle ear and mastoid cells in COM,        quantitatively, and hence to determine the grade of findings
in terms of histopathological and SEM findings. In refrac-        of infection and recovery. We hope that the present study
tory COM, in spite of medical treatment to which primary          will be a pioneer model that can guide future studies with
pathogen, P. aeruginosa, is sensitive, otorrhoea and inflam-      biofilms and resistant infections. In future studies, lower mi-
mation cannot be improved. NAC also exerts an eradicat-           croorganism concentrations can be used in conjunction with
ing effect on biofilm layers produced by various bacteria         higher dosages of NAC. This method may better reveal the
in addition to its other benefits 9-12,16,17. The effect of NAC   efficacy of NAC in the process of biofilm formation in COM.



                                                                                                                                    461
E. Ersoy Çallıoğlu et al.




Conclusions                                                                     9
                                                                                     Perez-Giraldo C, Rodriguez-Benito A, Moran FJ, et al. Influence
                                                                                     of N-acetylcysteine on the formation of biofilm by Staphylococcus
Although there are many studies demonstrating the effect                             epidermidis. J Antimicrob Chemother 1997;39:643-6. https://doi.
                                                                                     org/10.1093/jac/39.5.643
of NAC on biofilms, very few of them examined its effi-
                                                                                10
                                                                                     Marchese A, Bozzolasco M, Gualco L, et al. Effect of fosfomycin
cacy in COM. In our study, we found no additional effect                             alone and in combination with N-acetylcysteine on E. coli biofilms.
of topical NAC to the treatment of COM evaluated histo-                              Int J Antimicrob Agents 2003;22:95-100. https://doi.org/10.1016/
pathologically and with SEM in the rat model. We suggest                             s0924-8579(03)00232-2
that further studies are required using different concentra-                    11
                                                                                     Schwandt LQ, Van Weissenbruch R, Stokroos I, et al. Prevention of
tions of NAC with different experimental animals in order                            biofilm formation by dairy products and N-acetylcysteine on voice
                                                                                     prostheses in an artificial throat. Acta Otolaryngol 2004;124:726-31.
to reach more definitive conclusions.                                                https://doi.org/10.1080/00016480410022516
                                                                                12
                                                                                     Zhao T, Liu Y. N-acetylcysteine inhibit biofilms produced by Pseu-
References                                                                           domonas aeruginosa. BMC Microbiol 2010;10:140. https://doi.org/
                                                                                     10.1186/1471-2180-10-140
1
    Leone S, Molinaro A, Alfieri F, et al. The biofilm matrix of Pseu-          13
                                                                                     Dohar JE, Alper CM, Rose EA, et al. Treatment of chronic sup-
    domonas sp. OX1 grown on phenol is mainly constituted by alginate                purative otitis media with topical ciprofloxacin. Ann Otol
    oligosaccharides. Carbohydr Res 2006;341:2456-61. https://doi.
                                                                                     Rhinol Laryngol 1998;107(10 Pt 1):865-71. https://doi.org/
    org/10.1016/j.carres.2006.06.011
                                                                                     10.1177/000348949810701010
2
    O’Toole GA, Kaplan, HB. Biofilm formation as microbial develop-             14
                                                                                     Alper CM, Dohar JE, Gulhan M, et al. Treatment of chronic sup-
    ment. Ann Rev Microbiol 2000;54:49-79. https://doi.org/10.1146/an-
                                                                                     purative otitis media with topical tobramycin and dexamethasone.
    nurev.micro.54.1.49
                                                                                     Arch Otolaryngol Head Neck Surg 2000;126:165-73. https://doi.org/
3
    Dohar JE, Hebda PA, Veeh R, et al. Mucosal biofilm formation on                  10.1001/archotol.126.2.165
    middle-ear mucosa in a nonhuman primate model of chronic sup-
    purative otitis media. Laryngoscope 2005;115:1469-72. https://doi.
                                                                                15
                                                                                     Saylam G, Tatar EC, Tatar I, et al. Association of adenoid surface
    org/10.1097/01.mlg.0000172036.82897.d4                                           biofilm formation and chronic otitis media with effusion. Arch Oto-
                                                                                     laryngol Head Neck Surg 2010;136:550-5. https://doi.org/10.1001/
4
    Dodd S, Dean O, Copolov DL, et al. N-acetylcysteine for antioxidant              archoto.2010.70
    therapy: pharmacology and clinical utility. Expert Opin Biol Ther
    2008;8:1955-62. https://doi.org/10.1517/14728220802517901
                                                                                16
                                                                                     El-Feky MA, El-Rehewy MS, Hassan MA, et al. Effect of ciprofloxa-
                                                                                     cin and N-acetylcysteine on bacterial adherence and biofilm forma-
5
    Berk M, Copolov DL, Dean O, et al. N-acetyl cysteine for depres-                 tion on ureteral stent surfaces. Pol J Microbiol 2009;58:261-7.
    sive symptoms in bipolar disorder a double-blind randomized place-
    bo-controlled trial. Biol Psychiatry 2008;64:468-75. https://doi.org/
                                                                                17
                                                                                     Onger ME, Gocer H, Emir D, et al. N-acetylcysteine eradicates Pseu-
    10.1016/j.biopsych.2008.04.022                                                   domonas aeruginosa biofilms in bone cement. Scanning 2016;38:766-
                                                                                     70. https://doi.org/10.1002/sca.21326
6
    Riise GC, Qvarfordt I, Larsson S, et al. Inhibitory effect of N-acetyl-
    cysteine on adherence of Streptococcus pneumoniae and Haemophi-             18
                                                                                     Ozcan C, Görür K, Cinel L, et al. The inhibitory effect of topical
    lus influenzae to human oropharyngeal epithelial cells in vitro. Respi-          N-acetylcysteine application on myringosclerosis in perforated rat
    ration 2000;67:552-8. https://doi.org/10.1159/000067473                          tympanic membrane. Int J Pediatr Otorhinolaryngol 2002;63:179-84.
7
    Stey C, Steurer J, Bachmann S, et al. The effect of oral N-acetylcysteine        https://doi.org/10.1016/s0165-5876(01)00640-1
    in chronic bronchitis: aquantitative systematic review. Eur Respir J        19
                                                                                     Yamada M, Tsukimura N, Ikeda T, et al. N-acetyl cysteine as an os-
    2000;16:253-62. https://doi.org/10.1034/j.1399-3003.2000.16b12.x                 teogenesis-enhancing molecule for bone regeneration. Biomaterials
8
    Kauffman CA. Quandary about treatment of aspergillomas per-                      2013;34:6147-56. https://doi.org/10.1016/j.biomaterials.2013.04.064
    sists.   Lancet      1996;347:1640.      https://doi.org/10.1016/s0140-     20
                                                                                     Choe WT, Murray MT, Stidham KR, et al. N-acetylcysteine as an
    6736(96)91481-6                                                                  adjunct for refractory ear infections. Otol Neurotol 2007;28:1022-5.




462
