Abstract
Bacterial biofilms are organised complex structures having polymicrobial nature in a single community, which provide protection to bacteria from antibiotics by various means. The aim of our study was to determine the prevalence of biofilm-forming bacteria in clinical isolates of acute and chronic rhinosinusitis (ARS and CRS) patients with sinonasal mucopurulence. To know the incidence of bacterial biofilms in patient with ARS and CRS, to study the microbiology of bacterial biofilms in ARS and CRS, to assess the role and effects of biofilm in ARS and CRS and to correlate the association between the formation of the biofilm and development of rhinosinusitis. This prospective study was carried out at a tertiary care centre in Eastern part of India, in which 60 patients were taken as sample size. All patients of rhinosinusitis between age-group of 10 to70 years, who came to our out-patient department, were taken for our study. Biofilm formation was observed in 50% cases and were absent in 50% cases of chronic rhinosinusitis in our study. 83.3% (50) of patients out of 60 patients got improved after treatment and recurrence was observed in only 16.6% (10) of patients. Recurrence was more at 3 months follow-up as compared to follow-up at 1 month, though it was not statistically significant. Though our study highlighted the incidence and role of biofilms in the development of chronic rhinosinusitis, but few more randomized controlled studies involving larger sample sizes should be done to exactly determine the pathophysiological role of biofilms in the development and recurrence of acute and chronic rhinosinusuitis.
Keywords: Biofilms, Bacterial colony, Recurrence, Acute rhinosinusitis, Chronic rhinosinusitis
Introduction
Biofilm consist of a complex and organized community of bacteria, having both biotic and abiotic surface, which have live bacteria within extruded exopolysaccharide matrix .This provides evasion of host defense and thus decreases antibiotic susceptibility. Also, it has ability to release plank tonic bacteria resulting in recurrent acute infections. Bacterial biofilms were hypothesized to contribute in the progression as well as persistence of acute and chronic rhinosinusitis (ARS and CRS) [1]. Biofilms complicate infection management by antibiotic resistance and inconsistency of culture result [2]. Biofilms are polymicrobial, which may be homogenous or heterogenous. They have multiple bacteria and/or fungal species in single community, so having multiple advantages like passive resistance, metabolic cooperation, byproduct influence, quorum sensing system, enlarged gene pool with more efficient DNA sharing and many others, which provide competitive advantage. These biofilms contain pseudomonas aeruginosa, staphylococcus aureus and some other gram positive and gram negative strain that have the ability to secrete polysaccharide exopolymer which forms glycocalyx matrix, a covering of biofilm [3]. Biofilms have some defence ability to unfavourable environment that provides antibiotic resistance. Few literatures identify biofilm structure on the mucosal surface of patient with ARS and CRS [4–6]. While some literature concludes that there was no biofilm present in diseased person and they are present on mucosal surface of healthy controls also [7–9]. This provides multifactorial pathophysiology of ARS and CRS. The biofilm formation starts, when planktonic bacteria coalesces and adhere to surface and they begin to form microcolonies, which eventually progresses to biofilm formation [10]. 90% of biofilms are composed of exopolysaccharide matrix [11]. When biofilms form on ciliated mucosal surface, then they have additional properties and are called ‘mucosal biofilm.’ [12].
Given the well- documented evidence of biofilms in ARS and CRS, the aim of this study is to determine the prevalence of biofilm-forming bacteria in clinical isolates of ARS and CRS patients with sinonasal mucopurulence and to characterize the clinical factors that may predispose these patients to biofilm formation.
Aims & Objectives
To know the incidence of bacterial biofilm in patient with ARS and CRS.
To study the microbiology of biofilms in ARS and CRS.
To assess the role and effects of biofilm in ARS and CRS.
To correlate between the formation of the biofilm and development of recurrence.
Materials and Methods
This prospective study was carried out in a tertiary care centre.
Sample size- 60 patients.
Inclusion Criteria
Patient willing to give written informed consent for surgery.
Age group between 10 to70 years.
Cases of both acute and chronic rhinosinusitis.
Both sexes were included.
Exclusion Criteria
Patient not giving consent for surgery.
Pregnant ladies.
Patient with malignancy.
Patient with chronic granulomatous lesions.
Patient < 10 years and > 70 years.
The date was collected, compiled on MS excel sheet.
Statistical analysis was done using SPSS 25.
Method of Data Collection
Detailed history with the sign and symptoms of CRS followed by local (anterior rhinoscopy, posterior rhinoscopy and diagnostic nasal endoscopy) and systemic examination, radiological scans and routine investigations.
Diagnostic Nasal Endoscopy
Samples were taken and collected from nasal discharge and polypoidal tissue ( in CRS).
Processing of Samples for Biofilms
The samples would be crushed, vortexed, properly mixed and then inoculated on Blood agar and Macconkey agar plate and incubated for 48 h. Following incubation, growth would be identified based on colony morphology and biochemical reaction according to standard protocol. Smear preparation would also be directly from Gram staining and preliminary identification of pathogenic organism. Fungi were grown on Sabouraud’s Dextrose Broth at 37 °C for 72 h. Conidia were harvested by flooding the surface of the agar plates with 5 ml normal saline containing 0.025% (v/v) Tween 20 and rocking gently. The conidial suspension is then recovered and dispensed into sterile glass containers. The conidia is then counted using a Neubauer haemocytometer and adjusted to the required concentration in Sabouraud’s Dextrose Broth. All procedures are carried out in a laminar flow cabinet.
Detection of Biofilm Formation
This was done by tissue culture plate method using a semi-quantitative adherence assay. Briefly, an overnight culture of aerobic organism grown in BHI at 37 °C would be diluted to 1:100 in BHI with 2% glucose. A total of 200 µl of these cell suspensions would then be transferred in a U bottomed 96-well microliter plate (Tar sons, India). Wells with sterile BHI alone will serve as negative control. Each strain would then be tested in triplicate. The plate would then be incubated aerobically at 37 °C for 24 h. Then the microtiter wells would be washed twice with phosphate – buffered saline (PBS) and dried. Adherent bacteria would be fixed with 95% ethanol and stained with 1% crystal solution for 15 min. The microplate would be washed, air dried and the optical density of each well would be measured at 450 nm using an automated ELISA reader. Strong biofilm producers would be considered in those with OD value > 0.5.
Wild-type Pseudomonas aeruginosa (PAO1), a biofilm-producing strain, was used as a positive control in each of the 96-well microtiter plates. The sad-36 strain of P. aeruginosa, carrying a mutation in the flagella K gene, is unable to form a complete flagellum, which is required for attachment to surfaces and was used as a negative control for each 96-well microtiter plate. Furthermore, the few strain of P. aeruginosa possesses a mutation in the type IV pili gene, which is responsible for bacterial auto-aggregation. Therefore, this mutant is able to adhere to surfaces and form a dispersed monolayer of cells but is unable to organize into microcolonies and thus, develop into a mature biofilm.
Categorization of Isolates Based on Biofilm Forming Capacity [13]
The following criteria were used for biofilm gradation in clinical isolates:-.
OD cut = OD avg of negative control + 3 × standard deviation (SD) of ODs of negative control.
OD ≤ OD cut = Non-biofilm-former (NBF).
OD cut < OD ≤ 2 × OD cut = Weak biofilm-former (WBF).
2 × OD cut < OD ≤ 4 × OD cut = Moderate biofilm-former (MBF).
OD > 4 × OD cut = Strong biofilm-former.
Follow Up
Patients were called for follow-up after one and three month respectively.
Results and Observations
Bacterial or fungal biofilm formation was observed in 50% cases and were absent in 50% cases of acute and chronic rhinosinusitis in our study. Majority of patients with acute and chronic rhinosinusitis (43.33%) presented to us in adult age-group i.e. between 21 and 40 years of age. Genders wise distribution was nearly equal i.e. 32(53.3%) cases of rhinosinusitis were females and 28(46.7%) were males. Most of the patients of acute and chronic rhinosinusitis presented with the symptoms of facial pain (73.3%), nasal obstruction/blockage (88.3%) and nasal or post-nasal discharge (73.3%). Cultures of 23 cases were found to be positive for Staphylococcus aureus species and 7 for Pseudomonas aeruginosa species. However no bacterial culture growth was seen in 30 cases (i.e. sample was sterile). Culture of 3 cases was found to be positive for Aspergillus species, 3 cases for Candida species, and 2 cases for Rhizopus species. However no fungal culture growth was seen in 52 cases (86.67%). In our study, around 50% patients had shown growth of either bacterial or fungal colony on culture, but 50% did not show any growth of micro-organisms. Around 83.3% (50) of patients out of 60 patients got improved after treatment, while recurrence was observed in only 16.6% (10) of patients in our study. Recurrence was more at 3 months follow-up as compared to follow-up at 1 month, though it was not statistically significant. Proportion of recurrence present was increased from 0% at 1 month to 12.5% after 3 months in case of biofilm formation negative patients, whereas it got increased from 5.6 to 19.4% during 1 month and 3 months follow-up in biofilm formation positive patients, respectively. Our study also showed that 87.5% patients got improved in biofilm negative patients, while recurrence was seen in remaining 12.5%, whereas in case of biofilm positive patients, recurrence was seen in 19.4% patients. So, our study indicated that biofilm formation led to more chances of recurrence during long follow-up, though it was not statistically significant, as the number of patients was not that big in our study.
Discussion
Rhinosinusitis is characterized by the presence of inflammation in the mucosal lining of both the nasal passage and paranasal sinuses. Acute rhinosinusitis is prevalent among 8–15% of individual at any given moment.
Morphologically, biofilms are characterized by a three-dimensional complex of bacteria enclosed in a self-produced extracellular matrix formed by polysaccharides, nucleic acids, proteins and extracellular. Biofilms are organized bacterial communities that may be homogeneous or heterogeneous. They play a significant role in the pathogenesis of chronic nasal sinusitis, chronic tonsillitis, chronic supportive otitis media and device related infections. Biofilm development in the nasal mucosa has been linked to the decrease expression of anti-bacterial peptides like lactoferrin and MUC-7 as well as reduced level of TOLL like receptors responsible for recognising gram-positive bacteria [14].
As mentioned in the literature, there is a significant increase in the levels of interferon gamma, granulocytes colony stimulating factor, macrophages inflammatory protein beta and neutrophils observed in the sinonasal mucosa. Within the content of biofilms, there is an observed augmentation in the production levels of interleukin-5, 6 and eosinophilic cationic protein, indicating an enhanced immune response [15]. The presence of osteolytic bone beneath biofilm infected mucosa triggers an upregulation in the production of inflammatory cytokines, thus potentially acting as a focal point for the accumulation and dissemination of these pro-inflammatory mediators [16]. Biofilms represent an adaptable phenotypic transition in prokaryotic life prevalent across diverse bacterial species. They serve as the primary mechanism for bacterial survival and propagation. Several retrospective and prospective studies have elucidated the co-relation between bacterial biofilms and heightened disease severity prior to surgery as well as the persistence of refractory disease following surgical intervention [17].
In patients with rhinosinusitis, our study unveiled that a significant proportion comprising of 43.33% fell within the adult age group, specifically between 21 and 40 years of age, while another 30% of patients were beyond 40 years of age. (Table 1) Khalil et al. [18] demonstrated similar demographic trends in his research with a median age of 29 years among sample size of 190 patients. Ethnically, the patient cohort predominantly identified were as white (76%) followed by African American(13%), Hispanic (6%), Asian (2%) and remaining 3% either specified others or opted not to disclose their ethnicity in study done by Khalil et al. [18]
Table 1.
Age Group distribution among Acute and Chronic Rhinosinusitis patients (n = 60)
| Age Group (years) | Number of patients | Percentage of Patients |
|---|---|---|
| ≤ 20 | 16 | 26.6% |
| 21–40 | 26 | 43.33% |
| 41–60 | 15 | 25.00% |
| > 60 | 3 | 05.00% |
*Patients > 70 years and < 10 years are excluded from the study
Comments Majority of patients with Rhinosinusitis (43.33%) presented at adult age i.e., between 21–40 years of age. 30.00% of patients were older than 40 years of age, while 16 patients were below 20 years of age-group
The present study indicates that the majority of rhinosinusitis patient exhibited various symptoms with facial pain prevalent at 73.3% (44 individuals), nasal obstruction/ blockage at 83.3% (53individuals) and nasal or postnasal discharge/purulence at 73.3% (44 individuals). Headache was reported in 66.7% (40 individuals) of patients, fatigue affected 70% (42 individuals), ear pain/ pressure/fullness were experienced by 56.7% (34 individuals) and cough was present in 50% (30 individuals) of cases. Additionally, dental pain was noted in 48.3% (29 individuals) of patients, fever (other than acute rhinosinusitis) in 46.7% (28), hyposmia /anosmia in 45% (27) and halitosis in 35% (21). Fever specifically associated with acute rhinosinusitis was reported by 11.7% (7) of patients. (Table 2) Out of total 60 patients, discharge was observed in 92.9% of males and 84.4% of females while scarring occurred in 3.6% males and 37.5% of females respectively. (Table 3) Singhal D et al. [19] in 2010 showed similar type of association for the facial pain, nasal/ postnasal discharge, ear pain and fever, but gender association was not statistically significant in his study also. After endoscopic sinus surgery, bacterial biofilm formation was detected in majority of chronic bacterial infections [11]. Additionally, sleep apnoea has been linked to varying levels of nasal inflammation and airway hyperreactivity, while smoking has been identified as a risk factor for respiratory tract infections [20]. Fokkens et al. [21] in 2007 have demonstrated that nasal steroids are commonly utilised as the primary treatment modality for the chronic rhinosinusitis with and without polyps. Psaltis et al. [22] in 2008 showed similar success rates after surgery. Thus, the efficacy of steroids in managing infective CRS particularly following FESS warrants further investigation. In our study, only topical medication administered prior to sample collection were recorded. In our study, the presence or absence of biofilm formation was not significantly associated with gender of the patient, as biofilm formation was observed in 64.29% (18) of males and 56.3%(18) of females. While it was absent in 35.71% (10) of males and 43.80% (14) of females, out of total 60 patients. Additionally besides biofilms, the presence of fungus was the only factor identified to have statistically significant association with an unfavourable outcome. Dlugaszewska et al. [23] in 2016 demonstrated that 76.67% (23/30) of patients who underwent FESS for chronic rhinosinusitis exhibited evidence of biofilms in tissues upon examination of Scanning Electron Microscope (SEM) micrographs. Ramadan et al. [24] in 2006 and Sanclement et al. [17] in 2005 collectively detected bacterial biofilms in 14 out 18 patients accounting for 78% of study population. Psaltis et al. [22] in 2008 revealed in their study a robust correlation between mucosal outcome and biofilm status, indicating a very strong association between these factors. In the study by Robinsons et al. [25] in 2005, the analysis revealed that there was no statistically significant relationship between presence of pus and Lund-McKay scores. The current research indicates that the majority of patient experienced improvement, when biofilm formation was absent. However in cases, where biofilm formation was present, recurrence occurred in only 5.6% of patients. Furthermore comparing the two follow-up periods at 1month and 3 months, the proportion of recurrence increased from 0 to 12.5% after 3 months for patients without biofilm formation, whereas it increased from 5.6 to 19.4% during the same period in patient with biofilm formation. (Tables 4 and 5) In a study conducted in 2005, Ferguson and Stolz [26] provided evidence indicating the presence of bacterial biofilm in two out of four patients diagnosed with chronic rhinosinusitis. Sanclement and colleagues [17] in 2005 reported a significant finding indicating the presence of biofilms in a substantial majority, specifically 80% of mucosal biopsy obtained from individuals diagnosed with CRS.
Table 2.
Association of major and minor symptoms in males and females with Acute and Chronic Rhinosinusitis (n = 60)
| Symptoms | Male | Female | Total (n = 60) | ||||||
|---|---|---|---|---|---|---|---|---|---|
| Absent | Present | Absent | Present | ||||||
| No | Percentage | No | Percentage | No | Percentage | No | Percentage | ||
| Facial pain/pressure/fullness | 9 | 32.1% | 19 | 67.9% | 7 | 21.9% | 25 | 78.1% | 100% |
| Nasal obstruction/ blockage | 3 | 10.7% | 25 | 89.3% | 4 | 12.5% | 28 | 87.5% | 100% |
| Nasal or postnasal discharge/purulence | 8 | 28.6% | 20 | 71.4% | 8 | 25.0% | 24 | 75.0% | 100% |
| Hyposmia/anosmia | 15 | 53.6% | 13 | 46.4% | 18 | 56.3% | 14 | 43.8% | 100% |
| Fever (in acute rhinosinusitis only) | 24 | 85.7% | 4 | 14.3% | 29 | 90.6% | 3 | 9.4% | 100% |
| Headaches | 9 | 32.1% | 19 | 67.9% | 11 | 34.4% | 21 | 65.6% | 100% |
| Fever (other than acute rhinosinusitis) | 11 | 39.3% | 17 | 60.7% | 21 | 65.6% | 11 | 34.4% | 100% |
| Halitosis | 21 | 75.0% | 7 | 25.0% | 18 | 56.3% | 14 | 43.8% | 100% |
| Fatigue | 9 | 32.1% | 19 | 67.9% | 9 | 28.1% | 23 | 71.9% | 100% |
| Dental pain | 12 | 42.9% | 16 | 57.1% | 19 | 59.4% | 13 | 40.6% | 100% |
| Cough | 16 | 57.1% | 12 | 42.9% | 14 | 43.8% | 18 | 56.3% | 100% |
| Ear pain/pressure/fullness | 14 | 50.0% | 14 | 50.0% | 2 | 37.5% | 20 | 62.5% | 100% |
Comments In our study, most of the patients of rhinosinusitis presented with the symptoms of Facial pain/Pressure/Fullness in 67.9% (19) males and 78.1% (25) females, Nasal obstruction in 89.3% (25) males and 87.5% (28) females, Nasal or Postnasal discharge / purulence in 71.4% (20) males and 75.0% (24) females, Hyposmia in 46.4% (13) males and 43.8% (14) females, fever (in acute rhinosinusitis only) in 14.3% (4) males and 9.4% (3) females, Headaches in 67.9% (19) males and 65.6% (21) females, fever (other than acute rhinosinusitis) in 60.7% (17) males and 34.4% (11) females, Halitosis in 25.0% (7) males and 43.8% (14) females, Fatigue in 67.9% (19) males and 71.9% (23) females, Dental pain in 57.1% (16) males and 40.6% (13) females, Cough in 42.9% (12) males and 56.3% (18) females, Ear pain / pressure / fullness in 50.0% (14) males and 62.5% (20) females
Table 3.
Association of DNE (Diagnostic Nasal Endoscopy) findings in males and females with Acute and Chronic Rhinosinusitis (n = 60)
| DNE Findings | Sex | Total | |||||||
|---|---|---|---|---|---|---|---|---|---|
| Male | Female | ||||||||
| Absent | Present | Absent | Present | ||||||
| No. | % | No. | % | No. | % | No. | % | ||
| Polyp | 11 | 39.3% | 17 | 60.7% | 10 | 31.3% | 22 | 68.8% | 100% |
| Oedema | 0 | 0.0% | 28 | 100.0% | 0 | 0.0% | 32 | 100.0% | 100% |
| Discharge | 2 | 7.1% | 26 | 92.9% | 5 | 15.6% | 27 | 84.4% | 100% |
| Scarring | 27 | 96.4% | 1 | 3.6% | 20 | 62.5% | 12 | 37.5% | 100% |
Comments Diagnostic Nasal endoscopy was suggestive of Polyps in 39.3% males and 68.8% females, Oedema in 100% males and females, Discharge in 92.9% males and 84.4% females, Scarring in 3.6% and 37.5% females, out of total 60 patients. Our study also showed that only scarring was significantly associated with gender of the patients (p value < 0.05)
Table 4.
Association of bacterial and fungal colony with Acute and Chronic Rhinosinusitis (n = 60)
| Bacterial Colony | Number | Percentage | Fungal Colony | Number | Percentage |
|---|---|---|---|---|---|
| Negative | 30 | 50.0 | Negative | 52 | 86.67 |
| Pseudomonas aeruginosa | 7 | 11.66 | Aspergillus Species | 3 | 5.0 |
| Staphlylococcus aureus | 23 | 38.33 | Candida Species | 3 | 5.0 |
| Rhizopus Species | 2 | 3.33 | |||
| Total | 60 | 100 | Total | 60 | 100 |
Comments Culture of 23 cases was found to be positive for Staphylococcus aureus species and 7 for Pseudomonas aeruginosa species. However no bacterial culture growth was seen in 30 cases (i.e., sample was sterile). Culture of 3 cases was found to be positive for Aspergillus species, 3 cases for Candida species, 2 for Rhizopus species. However no fungal culture growth was seen in 52 cases out of 60 cases. (i.e., samples were sterile)
Table 5.
Comparison between first follow-up and second follow-up
| Biofilm Formation | Follow up on 1 month | Follow up on 3 months | ||
|---|---|---|---|---|
| Pt improved | Recurrence present | Pt improved | Recurrence present | |
| Negative | 24 | 0 | 21 | 3 |
| 100.0% | 0.0% | 87.5% | 12.5% | |
| Positive | 34 | 2 | 29 | 7 |
| 94.4% | 5.6% | 80.6% | 19.4% | |
| Total | 58 | 2 | 50 | 10 |
| 96.7% | 3.3% | 83.3% | 16.7% | |
Comments Above comparative table shows that proportion of recurrence present was increased from 0.0% at 1 month to 12.5%, where biofilm formation was negative, whereas it was increased from 5.6–19.4%, where biofilm formation was positive, at 1 month and 3 months respectively
Conclusion
Acute and chronic Rhinosinusitis (ARS and CRS) entails a complex interplay of infectious, inflammatory and host factors, highlighting the multifactorial nature of this condition. The aetiology of ARS and CRS is not solely attributed to infection by a single pathogenic bacterium; rather, it involves a more complex interplay of various factors, including microbial, inflammatory, and host-related elements. While biofilm undoubtedly plays a substantial role, it is essential to explore and understand the parallel correlation between biofilm formation and other etiopathogenetic factors to comprehensively grasp the mechanisms underlying acute and chronic sinusitis.
Though, our study highlighted the incidence and role of biofilms in the development and recurrence of acute and chronic rhinosinusitis, but only 50% cases in our study were positive for biofilm formation, and recurrence rates in positive cases were not statistically significant even after 3 months of follow-up. So, more randomized controlled studies or meta-analysis of all the studies published in literature should be done to exactly determine the pathophysiological role of biofilms in the development and recurrence of acute and chronic rhinosinusuitis and to frame the proper management guidelines for reducing the recurrence rate of acute and chronic sinusitis.
Acknowledgements
We would like to acknowledge our patients for giving consent for participation. Also, our audiologist team helped us in providing the hearing reports for all our patients.
Author Contributions
All authors have contributed equally in the research work of our study.
Funding
Nil.
Data Availability
Patients coming to our hospital were included and the results were compiled.
Declarations
Ethical Approval
Our work involved the routine surgery being done in our department for a long time. So, ethical approval was exempted for our study by our institute. The authors assert that the research was conducted ethically in accordance with the World Medical Association Declaration of Helsinki. Also, proper consent has been taken from all the participants before including in the study.
Consent for Publication
All authors have given the consent for publication of our work.
Competing Interests
Nil.
Footnotes
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
References
- 1.Cohen M, Kofonow J, Nayak JV et al (2009) Biofilms in chronic rhinosinusitis: a review. Am J Rhinol Allergy 23:255–260. 10.2500/ajra.2009.23.3319) [DOI] [PubMed] [Google Scholar]
- 2.Potera C (1999) Forging a link between biofilms and disease. Science 283:1837–1839 [DOI] [PubMed] [Google Scholar]
- 3.Foreman A, Boase S, Psaltis A, Wormald PJ (2012) Role of bacterial and fungal biofilms in chronic rhinosinusitis. Curr Allergy Asthma Rep 12:127e135 [DOI] [PubMed] [Google Scholar]
- 4.Cryer J, Schipor I, Perloff JR, Palmer JN (2004) Evidence of bacterial biofilms in human chronic sinusitis. ORL J Otorhinolaryngol Relat Spec 66:155e158 [DOI] [PubMed] [Google Scholar]
- 5.Sanclement JA, Webster P, Thomas J, Ramadan HH (2005) Bacterial biofilms in surgical specimens of patients with chronic rhinosinusitis. Laryngoscope 115:578e582 [DOI] [PubMed] [Google Scholar]
- 6.Perloff JR, Palmer JN (2004) Evidence of bacterial biofilms on frontal recess stents in patients with chronic rhinosinusitis. Am J Rhinol 18:377e380 [PubMed] [Google Scholar]
- 7.Ramadan HH, Sanclement JA, Thomas JG (2005) Chronic rhinosinusitis and biofilms. Otolaryngol Head Neck Surg 132:414e417 [DOI] [PubMed] [Google Scholar]
- 8.Sanderson AR, Leid JG, Hunsaker D (2006) Bacterial biofilms on the sinus mucosa of human subjects with chronic rhinosinusitis. Laryngoscope 116:1121e1126 [DOI] [PubMed] [Google Scholar]
- 9.Healy DY, Leid JG, Sanderson AR, Hunsaker DH (2008) Biofilms with fungi in chronic rhinosinusitis. Otolaryngol Head Neck Surg 138:641e647 [DOI] [PubMed] [Google Scholar]
- 10.Palmer J (2006) Bacterial biofilms in chronic rhinosinusitis. Ann Otol Rhinol Laryngol Suppl 196:35–39 [DOI] [PubMed] [Google Scholar]
- 11.Costerton JW, Stewart PS, Greenberg EP (1999) Bacterial biofilms: a common cause of persistent infections. Science 284:1318–1322 [DOI] [PubMed] [Google Scholar]
- 12.Hall-Stoodley L, Hu FZ, Gieseke A et al (2006) Direct detection of bacterial biofilms on the middle-ear mucosa of children with chronic otitis media. JAMA 296:202–211 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Singh AK, Prakash P, Achra A, Singh GP, Das A, Singh RK (2017) Standardization and classification of in vitro biofilm formation by clinical isolates of Staphylococcus aureus. J Global Infect Dis 9:93–101 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Psaltis AJ, Wormald PJ, Ha KR, Tan LW (2008) Reduced levels of lactoferrin in biofilm-associated chronic rhinosinusitis. Laryngoscope 118:895e901 [DOI] [PubMed] [Google Scholar]
- 15.Arjomandi H, Gilde J, Zhu S et al (2013) Relationship of eosinophils and plasma cells to biofilm in chronic rhinosinusitis. Am J Rhinol Allergy 27:e85ee90 [DOI] [PubMed] [Google Scholar]
- 16.ND A (2014) Biofilms in chronic rhinosinusitis. In: CC C, GA I, ME G (eds) Diseases of the sinuses. Spring, New York, p 110 [Google Scholar]
- 17.Sanclement JA, Webster P, Thomas J et al (2005) Bacterial biofilms in surgical specimens of patients with chronic rhinosinusitis. Laryngoscope 115:578–582 [DOI] [PubMed] [Google Scholar]
- 18.Khalil AN, Prince AA, Steiger JD et al (2008) Prevalence of biofilm-forming bacteria in chronic rhinosinusitis. Am J Rhinol 22:239–245 [DOI] [PubMed] [Google Scholar]
- 19.Singhal D, Psaltis AJ, Foreman A, Wormald PJ (2010) The impact of biofilms on outcomes after endoscopic sinus surgery. Am J Rhinol Allergy 24:169–174 [DOI] [PubMed] [Google Scholar]
- 20.Sabato R, Guido P, Salerno FG, Resta O, Spanevello A, Barbaro MP (2006) Airway inflammation in patients affected by obstructive sleep apnea. Monaldi Arch Chest Dis 65:102–105 [DOI] [PubMed] [Google Scholar]
- 21.Fokkens W, Lund V, Mullol J (2007) EP3OS 2007: European position paper on rhinosinusitis and nasal polyps. A summary for otorhinolaryngologists. Rhinology 45:97–101 [PubMed] [Google Scholar]
- 22.Psaltis AJ, Weitzel EK, Ha KR, Wormald PJ (2008) The effect of bacterial biofilms on post-sinus surgical outcomes. Am J Rhinol 22:1–6 [DOI] [PubMed] [Google Scholar]
- 23.Dlugaszewska J et al The pathophysiological role of bacterial biofilms in chronic sinusitis. Eur Archives Oto-Rhino-Laryngology 2016, 273.8: 1989–1994 [DOI] [PMC free article] [PubMed]
- 24.Ramadan HH, Sanclement JA, Thomas JG (2005) Chronic rhinosinusitis and biofilms. Otolaryngol Head Neck Surg 132:414–417 [DOI] [PubMed] [Google Scholar]
- 25.Robinson B, Kent C (2005) Espresso reward learning, holds the dopamine: theoretical comment on Robinson [DOI] [PubMed]
- 26.Ferguson BJ, Stolz DB (2005) Demonstration of biofilm in human bacterial chronic rhinosinusitis. Am J Rhinol 19:452–457 [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
Patients coming to our hospital were included and the results were compiled.