Abstract
Purpose
The purpose of the study was to investigate the expression of bacterial biofilm (BF) for chronic rhinosinusitis (CRS) and to find out factors affecting BF expression and the influences on its prognosis.
Materials and Methods
All specimens were analyzed by a scanning electron microscope and bacterial cultivation from a panel of 93 patients with CRS, 20 with nasal septum deviation, and 17 with nasal bone fractures as the control group. Referring to the grade classification criteria from the sinusitis-specific questionnaire Sino-Nasal Outcome Test–20, patients were assessed preoperatively based on common clinical manifestations. Patients were followed up for condition improvement as assessed by visual analogue scale and nasal endoscopic examination.
Results
In the experimental group, among all the patients, 3 were lost (lost 3.2%). Bacterial biofilm was positive in 64 (71.1%) of 90 patients. Mucosal cilia were observed in varying degrees of injury. Bacterial culture was positive in 60 (66.7%) patients. In the control group, no BF was found and no bacterium was cultured. Bacterial biofilm expression was correlated with the bacterial culture. The BF(+) patients’ visual analogue scale scores 6 months and 1 year postoperation were lower than the BF(−) patients’ scores ( P < .05). The BF(+) patients’ Lund-Kennedy scores at 6 months and 1 year postoperation were higher than the BF(−) patients’ scores ( P < .05).
Conclusions
Bacterial biofilm is involved in the pathogenesis of CRS and is associated with the bacterial culture. Bacterial biofilm has a certain impact on patients’ prognosis.
1
Introduction
Chronic rhinosinusitis (CRS) is a common disease in otolaryngology. Its pathogenesis has not yet been fully uncovered. The widely accepted multifactorial theory is that infection (bacteria, fungi, viruses), ostiomeatal complex body anatomical abnormalities, hypersensitivity, mucosa mucociliary system dysfunction, and others that are similar contribute to the disease. In recent years, bacteriological studies of CRS reported a positive rate of 32.5% to 96.0% , indicating a certain relationship between CRS and the bacteria. Corresponding to planktonic bacteria, bacterial biofilm (BF) is differently formed during the growth of bacteria to meet the requirements of its environment. Bacterial biofilm is composed of extracellular polymers secreted by the bacteria and the bacteria mosaic inside, with special characteristics of low growth rate, strong antibiotic resistance, mutual gene transferability, and others. Some researchers have proposed that BF is the common cause of chronic persistent infection . This idea provides the theoretical basis for the study of BF in CRS. A document review of international articles shows that researchers from around the world have thoroughly studied the expression of BF in CRS, the relationship between expression and pathology, the impact of BF growth capacity on the prognosis of CRS, etc. In China, related reports are few, mainly carried out on the morphology of BF in CRS. Still lacking are separate large-sample clinical studies on the relative effect between BF and clinical features/surgical results . The goals of this study are to observe BF expression on CRS patients’ mucosa specimens and to discover which factors affect BF expression and the influence on prognosis.
2
Materials and methods
2.1
Patients
Among the 93 CRS patients who underwent endoscopic sinus surgery between August 2007 and December 2008 in the ENT at Jinhua Central Hospital, 68 were positive for nasal polyps. The sample group was made up of 62 men and 31 women; the patients’ mean age was 35.38 ± 15.29 years. Visual analogue scale (VAS) assessment was mild (0–3 points) in 4 cases, moderate (3–7 points) in 51 cases, and severe (7–10 points) in 38 cases. Course of disease lasted 3 to 6 months in 26 cases, 6 months to 1 year in 9 cases, 1 to 3 years in 18 cases, 3 to 5 years in 2 cases, and more than 5 years in 35 cases. Case enrollment criteria were as follows: ( a ) symptoms of nasal congestion, pyorrhea, headache, and others; ( b ) symptoms lasting more than 3 months and preoperative endoscopic examination showing various degrees of edema of the nasal mucosa or nasal polyp; ( c ) at least a high density in the ethmoid sinus and even maxillary sinus by sinus computed tomography; ( d ) postoperatively pathologically diagnosed with nasal polyps or chronic inflammation of sinus mucosa; and ( e ) no significant improvement after preoperative nasal steroid spray (>1 month) and short-term oral antibiotic treatment (<2 weeks). Case exclusion criteria were as follows: ( a ) history of immunodeficiency or diabetes, ( b ) history of bronchiectasis, ( c ) history of cystic fibrosis, and ( d ) history of cancer. In the experimental group, we lost contact with 3 CRS patients (3.2%) because of change of address, change of telephone number, or other reasons. The control group included 20 cases with a deviated nasal septum and 17 cases with nasal bone fractures. Among them, 28 were male and 9 were female. The average age was 30.38 ± 8.22 years. All cases in the control group excluded CRS by sinus computed tomography.
2.2
Sample collection
In the experimental group, ethmoid sinus mucosa specimens (about 0.8 cm × 0.8 cm in size) were separately collected aseptically during endoscopic sinus surgery. The first specimens were bedside inoculated in anaerobic blood agar plates, placed and sealed in Anaerocults (Merck, Darm-stadt, Germany) (containing indicator), placed in sterile sample containers, and sent to bacteriology laboratory within 30 minutes. The second specimens were cleaned with isotonic sodium chloride solution to remove blood and secretions on the surface, fixed in 3% glutaraldehyde for more than 24 hours, and sent for electron microscope examination. In the control group, uncinate process mucosa specimens were collected under endoscope and processed in the same manner. All patients were informed of the operations, and they all signed informed consent forms.
2.3
Scanning electron microscope examination
The electron microscopy department cut the samples to a size of 3 mm × 3 mm, rinsed them each 3 times with phosphate-buffered saline for 45 minutes (15 minutes each time), and then dehydrated them 2 times with successive immersions in increasingly concentrated dilutions of acetone. The diluted concentrations were 30%, 50%, 70%, 80%, 90%, 95%, and 100%. The specimens were soaked for 20 minutes in each concentration each time. To substitute with an isoamyl acetate dilution, specimens were immersed in a mixture of isoamyl acetate and acetone (1:1) and then in pure isoamyl acetate dilution, both for 20 minutes. All specimens were appropriately shaken. Specimens were dried in an HCP-2 carbon dioxide critical point dryer, stuck with double-sided tape on the specimen stage, vacuum evaporated by a BC-II ion sputtering equipment, investigated under a KYKY-1000B scanning electron microscope (SEM), and digitally photographed by a WD-5 online SEM image analysis system.
Previously published articles have shown that BF formation can be divided into 3 stages. In the adhesion stage, bacteria on the mucosal surface adhere, aggregate, and produce extracellular polymeric matrix. In the mature stage of BF formation, round or oval bodies form a mosaic inside the matrix, forming a tower-like structure. These bodies are 0.05 to 5 µ m in size and contain 3-dimensional complexes that build open water channels. Bacteria proliferation in the microenvironment is composed of the extracellular polymeric matrix and water channels. Low optical microscopy showed that the mucosa surface was covered with BFs and tower-like structures separated by water channels. In the detachment stage, the small round or oval bodies are able to leave the matrix and plant themselves in a new area. Based on theories mentioned above, BFs of mucous membrane specimens of this experiment were observed.
2.4
Bacterial culture
The specimens were inoculated on 5% sheep blood agar plates, MacConkey agar plates, and Haemophilus / Neisseria filter plates (HAE 2 /BIO MERIEUX, Craponne, France); they were incubated at 35°C at 5% CO 2 for 18 to 24 hours and observed for growth of colonies. Anaerocults were also incubated at 35°C at 5% CO 2 for 48 hours and then observed. Following the Referring National Guide To Clinical Laboratory Procedures, separated suspected pathogens were Gram stained and underwent the catalase test or oxidase test. They were then identified either with a VITEK-AMS system (GPI card for gram positive, GNI + card for gram negative, and the ANI card for anaerobic bacteria) or an APINH system (for Neisseria / Haemophilus ). All bacteria were identified according to species.
2.5
Factors assessment
According to patients’ common clinical manifestations, a factors assessment scale was made to grade and classify criteria from the Sino-Nasal Outcome Test–20 (SNOT-20) . A staff member was especially assigned to communicate with patients in the experimental group on contents of the scale before the operation began. Because of the arrangement, the patients were able to comprehend our actions, which allowed us to complete our scale and achieve cooperation from the patients.
The factors on the assessment scale included entries on nasal congestion, runny nose, snot stench, headache, nasal discharge with blood, and hyposmia. Severity of snot stench was graded into the following categories: nonsmelly, a bit smelly, smelly, and very smelly. Severity of hyposmia was graded by patients’ ability to distinguish water, vinegar, and alcohol by smell, and was classified into the following categories: can distinguish all, can distinguish 2, can distinguish 1, and can distinguish none. The other 4 symptoms were graded as follows: no, occasionally, often, and always. All symptoms were given a score of 1 to 4 according to severity. In detail, 1 meant light and 4 meant most severe.
2.6
Postoperative follow-up
Postoperative follow-up started with conventional reexamination, cleaning of the surgical cavity, and debridement of crusts every week after discharge. In the second and third months, patients were reexamined once every 2 weeks. Vesicles and granulation in the surgical cavity were débrided, and adhesions were separated. Monthly examinations began from the fourth month after discharge. Appropriate additional examinations were needed if any patient experienced relapse. Prognosis evaluation was performed 6 months and 1 year after operation.
2.7
Subjective evaluation
Improvement of symptoms in patients was quantitatively assessed by VAS, scored from 0 to 10. In this scale, 0 = no symptoms improvement, whereas 10 = no symptoms were experienced. The VAS chart surface was hidden from patients. After the standards were fully explained to the patients and were understood, the patients scored themselves according to the degree of improvement of their condition.
2.8
Objective evaluation
Symptoms were objectively evaluated by endoscopic examination using the Lund-Kennedy (L-K) score .
2.9
Quality control
One researcher was appointed to evaluate the completed questionnaire, and this same researcher was in charge of explaining the purposes of the survey to the patients in detail and stressing the necessary importance that the questionnaire should be filled out as truthfully as possible. Two experienced surgeons performed surgeries. After operation, we gave the patients cephalosporin antibiotics for 2 weeks (for bacterial culture-positive patients, course was adjusted according to antibiotic sensitivity test), mucus decorporating agent for 2 weeks, and budesonide nasal spray for 8 to 12 weeks. The same group implemented the standardized treatment and follow-up. Prognosis evaluation was assigned to one researcher who was responsible for explaining the purpose of the survey to the patients in detail and emphasizing the importance of them giving authentic answers. Data were separately input and checked by 2 staff members. The blind method was used for questionnaire evaluation and prognosis assessment.
2.10
Statistical process
All analyses were made with the assistance of SPSS (Chicago, IL) 10 statistical software. The impacts of sex, disease severity, type, duration, and symptoms such as nasal congestion, runny nose, snot stench, headache, nasal discharge with blood, and hyposmia on formation of BF were analyzed through χ 2 test. By Fisher test, BF was analyzed in association with the bacterial culture. Results of follow-up data (by VAS score and L-K score) were expressed in mean ± standard deviation (SD). t test analyzed the impact of BF expression on prognosis. P < .05 was considered statistically significant in all the analyses.
2
Materials and methods
2.1
Patients
Among the 93 CRS patients who underwent endoscopic sinus surgery between August 2007 and December 2008 in the ENT at Jinhua Central Hospital, 68 were positive for nasal polyps. The sample group was made up of 62 men and 31 women; the patients’ mean age was 35.38 ± 15.29 years. Visual analogue scale (VAS) assessment was mild (0–3 points) in 4 cases, moderate (3–7 points) in 51 cases, and severe (7–10 points) in 38 cases. Course of disease lasted 3 to 6 months in 26 cases, 6 months to 1 year in 9 cases, 1 to 3 years in 18 cases, 3 to 5 years in 2 cases, and more than 5 years in 35 cases. Case enrollment criteria were as follows: ( a ) symptoms of nasal congestion, pyorrhea, headache, and others; ( b ) symptoms lasting more than 3 months and preoperative endoscopic examination showing various degrees of edema of the nasal mucosa or nasal polyp; ( c ) at least a high density in the ethmoid sinus and even maxillary sinus by sinus computed tomography; ( d ) postoperatively pathologically diagnosed with nasal polyps or chronic inflammation of sinus mucosa; and ( e ) no significant improvement after preoperative nasal steroid spray (>1 month) and short-term oral antibiotic treatment (<2 weeks). Case exclusion criteria were as follows: ( a ) history of immunodeficiency or diabetes, ( b ) history of bronchiectasis, ( c ) history of cystic fibrosis, and ( d ) history of cancer. In the experimental group, we lost contact with 3 CRS patients (3.2%) because of change of address, change of telephone number, or other reasons. The control group included 20 cases with a deviated nasal septum and 17 cases with nasal bone fractures. Among them, 28 were male and 9 were female. The average age was 30.38 ± 8.22 years. All cases in the control group excluded CRS by sinus computed tomography.
2.2
Sample collection
In the experimental group, ethmoid sinus mucosa specimens (about 0.8 cm × 0.8 cm in size) were separately collected aseptically during endoscopic sinus surgery. The first specimens were bedside inoculated in anaerobic blood agar plates, placed and sealed in Anaerocults (Merck, Darm-stadt, Germany) (containing indicator), placed in sterile sample containers, and sent to bacteriology laboratory within 30 minutes. The second specimens were cleaned with isotonic sodium chloride solution to remove blood and secretions on the surface, fixed in 3% glutaraldehyde for more than 24 hours, and sent for electron microscope examination. In the control group, uncinate process mucosa specimens were collected under endoscope and processed in the same manner. All patients were informed of the operations, and they all signed informed consent forms.
2.3
Scanning electron microscope examination
The electron microscopy department cut the samples to a size of 3 mm × 3 mm, rinsed them each 3 times with phosphate-buffered saline for 45 minutes (15 minutes each time), and then dehydrated them 2 times with successive immersions in increasingly concentrated dilutions of acetone. The diluted concentrations were 30%, 50%, 70%, 80%, 90%, 95%, and 100%. The specimens were soaked for 20 minutes in each concentration each time. To substitute with an isoamyl acetate dilution, specimens were immersed in a mixture of isoamyl acetate and acetone (1:1) and then in pure isoamyl acetate dilution, both for 20 minutes. All specimens were appropriately shaken. Specimens were dried in an HCP-2 carbon dioxide critical point dryer, stuck with double-sided tape on the specimen stage, vacuum evaporated by a BC-II ion sputtering equipment, investigated under a KYKY-1000B scanning electron microscope (SEM), and digitally photographed by a WD-5 online SEM image analysis system.
Previously published articles have shown that BF formation can be divided into 3 stages. In the adhesion stage, bacteria on the mucosal surface adhere, aggregate, and produce extracellular polymeric matrix. In the mature stage of BF formation, round or oval bodies form a mosaic inside the matrix, forming a tower-like structure. These bodies are 0.05 to 5 µ m in size and contain 3-dimensional complexes that build open water channels. Bacteria proliferation in the microenvironment is composed of the extracellular polymeric matrix and water channels. Low optical microscopy showed that the mucosa surface was covered with BFs and tower-like structures separated by water channels. In the detachment stage, the small round or oval bodies are able to leave the matrix and plant themselves in a new area. Based on theories mentioned above, BFs of mucous membrane specimens of this experiment were observed.
2.4
Bacterial culture
The specimens were inoculated on 5% sheep blood agar plates, MacConkey agar plates, and Haemophilus / Neisseria filter plates (HAE 2 /BIO MERIEUX, Craponne, France); they were incubated at 35°C at 5% CO 2 for 18 to 24 hours and observed for growth of colonies. Anaerocults were also incubated at 35°C at 5% CO 2 for 48 hours and then observed. Following the Referring National Guide To Clinical Laboratory Procedures, separated suspected pathogens were Gram stained and underwent the catalase test or oxidase test. They were then identified either with a VITEK-AMS system (GPI card for gram positive, GNI + card for gram negative, and the ANI card for anaerobic bacteria) or an APINH system (for Neisseria / Haemophilus ). All bacteria were identified according to species.
2.5
Factors assessment
According to patients’ common clinical manifestations, a factors assessment scale was made to grade and classify criteria from the Sino-Nasal Outcome Test–20 (SNOT-20) . A staff member was especially assigned to communicate with patients in the experimental group on contents of the scale before the operation began. Because of the arrangement, the patients were able to comprehend our actions, which allowed us to complete our scale and achieve cooperation from the patients.
The factors on the assessment scale included entries on nasal congestion, runny nose, snot stench, headache, nasal discharge with blood, and hyposmia. Severity of snot stench was graded into the following categories: nonsmelly, a bit smelly, smelly, and very smelly. Severity of hyposmia was graded by patients’ ability to distinguish water, vinegar, and alcohol by smell, and was classified into the following categories: can distinguish all, can distinguish 2, can distinguish 1, and can distinguish none. The other 4 symptoms were graded as follows: no, occasionally, often, and always. All symptoms were given a score of 1 to 4 according to severity. In detail, 1 meant light and 4 meant most severe.
2.6
Postoperative follow-up
Postoperative follow-up started with conventional reexamination, cleaning of the surgical cavity, and debridement of crusts every week after discharge. In the second and third months, patients were reexamined once every 2 weeks. Vesicles and granulation in the surgical cavity were débrided, and adhesions were separated. Monthly examinations began from the fourth month after discharge. Appropriate additional examinations were needed if any patient experienced relapse. Prognosis evaluation was performed 6 months and 1 year after operation.
2.7
Subjective evaluation
Improvement of symptoms in patients was quantitatively assessed by VAS, scored from 0 to 10. In this scale, 0 = no symptoms improvement, whereas 10 = no symptoms were experienced. The VAS chart surface was hidden from patients. After the standards were fully explained to the patients and were understood, the patients scored themselves according to the degree of improvement of their condition.
2.8
Objective evaluation
Symptoms were objectively evaluated by endoscopic examination using the Lund-Kennedy (L-K) score .
2.9
Quality control
One researcher was appointed to evaluate the completed questionnaire, and this same researcher was in charge of explaining the purposes of the survey to the patients in detail and stressing the necessary importance that the questionnaire should be filled out as truthfully as possible. Two experienced surgeons performed surgeries. After operation, we gave the patients cephalosporin antibiotics for 2 weeks (for bacterial culture-positive patients, course was adjusted according to antibiotic sensitivity test), mucus decorporating agent for 2 weeks, and budesonide nasal spray for 8 to 12 weeks. The same group implemented the standardized treatment and follow-up. Prognosis evaluation was assigned to one researcher who was responsible for explaining the purpose of the survey to the patients in detail and emphasizing the importance of them giving authentic answers. Data were separately input and checked by 2 staff members. The blind method was used for questionnaire evaluation and prognosis assessment.
2.10
Statistical process
All analyses were made with the assistance of SPSS (Chicago, IL) 10 statistical software. The impacts of sex, disease severity, type, duration, and symptoms such as nasal congestion, runny nose, snot stench, headache, nasal discharge with blood, and hyposmia on formation of BF were analyzed through χ 2 test. By Fisher test, BF was analyzed in association with the bacterial culture. Results of follow-up data (by VAS score and L-K score) were expressed in mean ± standard deviation (SD). t test analyzed the impact of BF expression on prognosis. P < .05 was considered statistically significant in all the analyses.
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