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Bacterial biofilms in chronic rhinosinusitis and their relationship with inflammation severity
Auris Nasus Larynx 39 (2012) 169–174 www.elsevier.com/locate/anl
Bacterial biofilms in chronic rhinosinusitis and their relationship with inflammation severity Hai-Hong Chen a, Xiang Liu a, Chao Ni a, Yin-Ping Lu a, Gao-Yun Xiong b, Yu-Yu Lu a, Shen-Qing Wang a,* a
Department of Head-Neck Otolaryngology, The First Affiliated Hospital, College of Medicine, Zhejiang University, Zhejiang Province, Hangzhou 310003, China b Department of Head-Neck Otolaryngology, TongDe Hospital of Zhejiang Province, Hangzhou 310013, China Received 23 December 2010; accepted 27 April 2011
Abstract Aims: To identify the presence of bacterial biofilms on mucosal specimens from chronic rhinosinusitis (CRS) patients, and evaluate their relationship with severity of CRS. Methods: A prospective study of biofilms presence on 24 CRS patients compared with 12 controls was designed. The presence of biofilms was determined by scanning electron microscopy (SEM), and associations with the preoperative Lund–MacKay CT scores, Johansson endoscopic scores, and the history of ESS were assessed. Results: Biofilms were found in 13/24 CRS patients (54.2%) but in only 1/12 controls (8.3%; P < 0.01). CRS patients with and without biofilms had similar preoperative Lund–MacKay CT and Johansson endoscopic scores (P > 0.05). Patients with revision ESS showed a tendency of higher biofilms incidence (5/7, 71.4%) than those undergoing their first procedure (8/17, 47.1%), but did not reach a significant difference (P > 0.05). Conclusions: The higher incidence of biofilms in CRS patients suggests a role in the pathogenesis of CRS, but no correlation with severity of CRS. # 2011 Elsevier Ireland Ltd. All rights reserved. Keywords: Bacterial biofilms; Chronic rhinosinusitis; Lund–MacKay CT scores; Johansson endoscopic scores; History of endoscopic sinus surgery
1. Introduction Bacterial biofilms are highly organized structures composed of bacterial communities encased within a protective extracellular matrix, which are resistant to both antibiotic treatment and host defense systems [1]. Biofilms are considered a common and important cause of persistent infections. The National Institutes of Health has estimated that at least 65% of all bacterial infections in humans are related to biofilms [2]. In the field of otolaryngology, biofilms have been documented on otitis media with effusion [3], cholesteatoma and tonsillitis [4,5], rhinosinusitis and * Corresponding author. Tel.: +86 571 87236895. E-mail address: [email protected] (S.-Q. Wang).
adenoids removed from children with chronic rhinosinusitis (CRS) [6], and they have also been isolated on some prosthetic devices, such as tracheotomy and tympanostomy tubes, front recess stents, and cochlear implants [7]. Current research suggests that biofilms may contribute greatly to the recalcitrant nature of CRS. There have been several reports regarding the presence of biofilms in surgical specimens obtained from patients with CRS. In 2005, Ramadan examined five specimens from five CRS patients, all of which were positive for biofilms [8]. Using confocal scanning laser microscopy, Psaltis found biofilms formation in 44% of 38 CRS patients [9]. A recent prospective study in over 150 consecutive CRS patients identified a positive rate of almost 30% for bacteria with biofilm-forming capacity, as assessed by an in vitro biofilm-formation assay [10].
0385-8146/$ – see front matter # 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.anl.2011.04.014
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H.-H. Chen et al. / Auris Nasus Larynx 39 (2012) 169–174
Although the association between biofilms and the recalcitrant nature of CRS has been gradually accepted by most researchers, the precise role of biofilms in the pathogenesis of CRS remains unclear. Additionally, the pathogenesis of CRS may differ between different populations and areas. There are only a few reports regarding the pathogenesis of CRS with biofilms in China, and even fewer concerning the clinical relevance of CRS with biofilms. The primary aim of this study was to determine the presence of biofilms on CRS patients in Chinese community and its correlations with Lund–Mackay CT scores, Johansson endoscopic scores, and history of endoscopic sinus surgery (ESS).
2. Materials and methods 2.1. Study design and population This was a prospective study performed to determine the presence of bacterial biofilms on the sinus mucosa of 24 CRS patients undergoing ESS compared with 12 controls without CRS. All patients were from the Otolaryngological Department of the 1st Affiliated Hospital of Zhejiang University and Zhejiang Provincial Tongde Hospital during the period July 2008 to September 2010. All subjects gave informed consent prior to enrollment in the study. All CRS patients fulfilled the diagnostic criteria for CRS according to clinical practice guidelines [11]. Clinical data, including demographic information, presence of bronchial asthma, macrolide therapy or steroid therapy before surgery, preoperative Lund–MacKay computed tomography scores, Johansson endoscopic scores and history of ESS, were recorded. Specimens were obtained intraoperatively from diseased sinuses, including the uncinate process, anterior ethmoid sinuses, posterior ethmoid sinuses or maxillary sinus, as evidenced by the preoperative CT scan. Multiple specimens were obtained per subject (47 specimens in all from 24 CRS patients), each of them varied from 3.0 mm 3.0 mm to 6.0 mm 6.0 mm. Specimens were washed thoroughly in saline, and then placed immediately into 2% glutaraldehyde for scanning electron microscopy (SEM) specimen preparation. The control group consisted of 12 patients without CRS (1 patient with nasopharyngeal carcinoma, 8 patients scheduled for nasal bone-plasty due to nasal trauma, 3 patients undergoing septoplasty). Specimens were obtained from the uncinate process or the anterior ethmoid bulla at the time of surgery.
using PBS. Next, the specimens were fixed with 1% osmium tetroxide for 1 h. They were then dehydrated through a graded ethanol series as follows: 50% for 15 min, 70% for 15 min, 80% for 15 min, 90% for 15 min, and 100% twice for 15 min each time. The tissue was immersed in 100% acetone for 15 min and washed in 100% isoamyl acetate for 15 min, followed by critical point drying. Finally, specimens were mounted on metal stubs and subsequently sputtercoated with gold preparation for imaging. Observations were carried out in our SEM laboratory using a Stereoscan 260 microscope (Leica, Wetzlar, Germany) at an acceleration voltage of 15 kV. Structures characterized by water channels, 3D structure, and matrixembedded spherical or elliptical bodies were identified as evidence of biofilms. It is differed from viscous mucous, the latter is a flat blanket, under the blanket, sometimes you may see the comparative orderly cilia, above the mucous blanket, sometimes you can find irregular foreign granule. The entire area of each specimen was scanned for the presence of biofilms structures. Images were taken at various angles to effectively display the specimens and to minimize errors and artifacts. The investigators examining the samples were blinded to the disease status of the patients. 2.3. Statistical analysis The results of this study were analyzed using the SPSS software (ver. 13.0; SPSS Inc., Chicago, IL). Continuous data were analyzed using Student’s t-test, and dichotomous data were analyzed using Fisher’s exact test. In all analyses, P values <0.05 were deemed to indicate statistical significance.
3. Results 3.1. Patient demographics and clinical data Twenty-four CRS patients and 12 controls were included in the study. The CRS group consisted of 16 males and 8 females with a mean age of 41.88 12.58 years (range 18–65 years), while the control group consisted of 8 males and 4 females with a mean age of 30.2 12.6 years (range 17–56 years). Table 1 lists the presence of bronchial asthma, macrolide therapy or steroid therapy before surgery, preoperative Lund–MacKay CT scores, Johansson endoscopic scores, and the history of ESS for each CRS patient.
2.2. Scanning electronic microscopy
3.2. SEM findings
All specimens were prepared for SEM using the following techniques. Tissue was initially fixed for 2 h in 2.5% glutaraldehyde in phosphate-buffered saline (PBS, pH 7.4) at 4 8C. Two rinses of 15 min each were then carried out
Specimens from the 24 CRS patients and 12 controls were examined for the presence of bacterial biofilms by SEM. Using strict SEM morphological criteria as well as the characteristic features of biofilms, all 24 CRS patients
H.-H. Chen et al. / Auris Nasus Larynx 39 (2012) 169–174
171
Table 1 Clinical summary and the presence of biofilms in the CRS group identified by SEM. Patients
Genderb/agea
No. No. No. No. No. No. No. No. No. No. No. No. No. No. No. No. No. No. No. No. No. No. No. No.
M/31 M/57 F/55 M/37 F/18 M/56 M/44 M/34 M/30 M/60 F/40 M/33 F/21 M/50 M/44 F/40 F/54 M/48 F/54 M/39 M/51 M/44 F/25 M/28
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
Bronchial asthma
Macrolide therapy
Steroid therapy
Previous ESS
Rhinocort (3Ms) Rhinocort (11Ms)
1 2 2 1
+(10Ys)
Rhinocort (6Ms) +(10Ys) 1 +(13Ys) +(15Ys) Rhinocort (1W)
2 1
Clarithromycin (1M)
Rhinocort (1M) Flisonase (2.5Ms)
Lund–MacKay CT scorec
Johansson endoscopic scorec
11 12 10 10 12 2 6 9 6 8 6 9 2 11 12 6 9 2 11 7 8 6 4 3
3 6 6 3 5 4 4 4 4 3 3 5 1 4 3 3 3 6 6 1 3 4 2 3
Biofilms presence + + + +
+ + + + + + + +
+
ESS: endoscopic sinus surgery; SEM: scanning electron microscopy; (+) positive; ( ) negative; n: number of previous ESS; Y: year; W: week; M: month. a In years. b M – male, F – female. c Scores of unilateral side (right or left) from which specimens taken.
showed aberrant findings on the mucosal surface with varying degrees of severity, from disarrayed cilia to the complete absence of cilia and goblet cells (Fig. 1). Characteristic morphological features of biofilms, such as water channels, 3D structure, and matrix-embedded spherical or elliptical bodies, were noted in 21 specimens from 13 cases of 24 CRS patients (54.2%, Table 1), the typical figures are showed in Fig. 2. Only 1 of the 12 controls (8.3%) showed evidence of biofilm; in the remaining 11
cases, though disarray and absence of cilia can also be found in scattered regions, the majority area of each specimen showed apparently healthy cilia and goblet cells as well as mucus blanket coating mucosa (Fig. 3), The differences in incidence rates of biofilms between CRS patients and the controls was statistically significant (Fisher’s exact test, P = 0.0079).
3.3. Correlation between the presence of bacterial biofilms and the severity of Lund–MacKay CT scores, Johansson endoscopic scores, and ESS history
Fig. 1. SEM showing denuded epithelium and scarce, disarrayed cilia in sinus mucosa of CRS patient.
Table 2 shows a comparison of the preoperative Lund– MacKay CT scores and Johansson endoscopic scores between CRS patients with and without biofilms. There was no identifiable differences in preoperative Lund– MacKay CT scores or Johansson endoscopic scores between patients with and without biofilms (Table 2). With regard to the history of ESS, among the seven CRS patients who had undergone previous ESS procedures, five were positive for biofilms. The incidence of biofilms seemed higher in patients with revision ESS (5/7, 71.4%) than in those undergoing their first procedure (8/17, 47.1%), but the difference was not statistically significant (Fisher’s exact test, P = 0.205).
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Fig. 2. SEM micrograph of biofilms in chronic rhinosinusitis patients: (A) showing 3D structure with water channels and (B) showing spherical bodies embedded in matrix.
Fig. 3. SEM appearance of a healthy control specimen showing (A) healthy epithelium and cilia and (B) a layer of mucous blanket above the cilia, with some foreign granule on the surface.
Table 2 Comparison CT and endoscopic findings between CRS patients with and without biofilm formation.
Lund–MacKay CT score Johansson endoscopic score *
Biofilm visualized (n = 13)
No biofilm visualized (n = 11)
P-value *
7.538 3.688 3.769 1.536
7.500 3.100 3.636 1.362
0.9443 0.8262
Student’s t-test.
4. Discussion Biofilms are increasingly recognized as playing an etiological role in CRS. Bacteria in biofilms are difficult to culture and largely resistant to host defense systems and antimicrobial therapy. The cultures of the planktonic bacteria in nasal discharge did not correlate with those in biofilms [12]. In fact, bacteria can be examined in normal nasal cavity, just as indicated by the study of Sanclement ‘‘All swabbed specimens, including controls, grew bacteria on routine cultures’’ [13]. In our prior study, we detected bacteria in nasal discharge in a group of 31 CRS patients, most of them were Staphylococcus epidermidis, others including Staphylococcus aureus, Pseudomonas aeruginosa, Enterobacter aerogenes, Streptococcus viridians, Staphylococcus haemo-
lyticus, Klebsiella pneumoniae, and Proteus vulgaris. But these may not quite the same as what within the biofilms, so in this study, we have not completed bacteria culture in nasal discharge. Usually the pathogens in biofilms can be exactly detected by fluorescence in situ hybridization (FISH). According to literatures, the predominant organisms were Staphylococcus aureus, Haemophilus influenzae, others including Pseudomonas aeruginosa, Streptococcus pneumophilia, and fungal elements could also be identified [12,14,15]. The incidence of biofilms in our CRS group was 54.2%, which was slightly lower than those in most previous studies. Sanclement et al. reported biofilms in 24 of 30 (80%) CRS patients using scanning electron microscopy [13]. Using confocal scanning laser microscopy (CSLM) together with
H.-H. Chen et al. / Auris Nasus Larynx 39 (2012) 169–174
BacLight staining or fluorescence in situ hybridization (FISH), Foreman et al. found biofilms in 18 of 20 (90%) CRS patients [16]. The reported incidence rates of biofilms in CRS patients range from 25% to 100% [9,12,13,16–19], with most studies showing rates of 70–80% [9,12,13,17]. This may be due to different populations, selection of materials for testing which represent only a small fraction of the total sinus mucosa, and also the technique used. CSLM and FISH usually have higher sensitivity and specificity than SEM, because they allow not only nondestructive analysis of the living biofilms, but also yield more detailed information on their entire structure [16]. However, regardless of the technique used, almost all previous studies demonstrated a significantly higher incidence of biofilms in CRS patients than in controls, suggesting that biofilms may play an active role in the pathogenesis of CRS. In the present study, all of the samples from the CRS group showed aberrant findings on the mucosal surface, with varying degrees of severity from disarrayed cilia to complete absence of cilia and goblet cells, even in patients without evidence of biofilms formation, while in the control group, the majority area of each specimen showed apparently normal epithelium and cilia. The mucociliary system can prevent initial bacterial approach. The disarray and absence of the mucociliary apparatus will facilitate biofilms development, and therefore reduction of sinonasal inflammation and protection of cilia represent the first step in blocking the attachment and aggregation of bacteria that would otherwise initiate biofilms formation. With regard to the correlation of biofilms presence with the severity of CRS, Singhal reported that the biofilmpositive group had significantly poorer Lund–McKay radiology and Kennedy–Lund endoscopic scores compared to those negative for biofilms, indicating more severe preoperative disease in this group of patients [17]. However, unlike the majority of reports published to date [9,17,20,21], our present study did not show a relationship between the presence of biofilms and the severities of preoperative Lund–MacKay CT and Johansson endoscopic scores between CRS patients with and without biofilms. These observations indicated that biofilms represent just one state in which bacteria exist, and biofilm-associated bacteria may have stronger resistance to both antibiotic treatment and host defense, resulting in recalcitrant infection. However, biofilms may not parallel the severity of inflammation, because the severity of CRS inflammation is multifactorial. It should be noted that the lack of correlation in the present study may have been due to the differences in study methods used, the patient population, and random specimen selection as well as subjective evaluation. Additionally, the cohort in the present study was small, and further studies with larger sample sizes are required. Although there was no statistically significant relationship between the presence of biofilms and a history of previous sinus surgery, patients with revision ESS tended to show a higher rate of biofilms than those undergoing their
173
primary surgery, which in agreement with findings reported previously by Psaltis and Singhal [9,17]. Due to the small number of cases with a history of previous sinus surgery in the present study, further studies with larger sample sizes are needed. Prince reported a significant correlation between the presence of biofilms and the number of prior ESS procedures [10], which suggested that biofilms may contribute to the recurrence of CRS and result in unfavorable outcomes of ESS as residual biofilms left behind after surgery may serve as a nidus for bacterial microcolony and biofilm regeneration, and thus act as stimuli for an ongoing inflammatory response. Although the presence of biofilms in CRS has been suggested previously, the present study added further evidence of the existence of biofilms in the Chinese community. The presence of biofilms may partially explain the recurrent nature of CRS and its refractoriness to surgical intervention. Further studies with larger sample sizes are required to assess the relationship between the presence of biofilms and the severity of CRS, as well as risk factors contributing to biofilms formation.
Conflict of interest None.
Acknowledgement Project Foundation: The Project Supported by Medical Scientific Research Foundation of Zhejiang Province, China (no. 2008B083).
References [1] Costerton JW, Stewart PS, Greenberg EP. Bacterial biofilms: a common cause of persistent infections. Science 1999;284:1318–26. [2] Potera C. Forging a link between biofilms and disease. Science 1999;283:1837–9. [3] Hall-Stoodley L, Hu FZ, Gieseke A, Nistico L, Nguyen D, Hayes J, et al. Direct detection of bacterial biofilms on the middle-ear mucosa of children with chronic otitis media. J Am Med Assoc 2006;296: 202–11. [4] Chole RA, Faddis BT. Evidence for microbial biofilms in cholesteatomas. Arch Otolaryngol Head Neck Surg 2002;128:1129–33. [5] Chole RA, Faddis BT. Anatomical evidence of microbial biofilms in tonsillar tissues: a possible mechanism to explain chronicity. Arch Otolaryngol Head Neck Surg 2003;129:634–6. [6] Zuliani G, Carron M, Gurrola J, Coleman C, Haupert M, Berk R, et al. Identification of adenoid biofilms in chronic rhinosinusitis. Int J Pediatr Otorhinolaryngol 2006;70:1613–7. [7] Post JC, Hiller NL, Nistico L, Stoodley P, Ethlich GD. The role of biofilms in otolaryngologic infections: update 2007. Curr Opin Otolaryngol Head Neck Surg 2007;15:347–51. [8] Ramadan HH, Sanclement JA, Thomas JG. Chronic rhinosinusitis and biofilms. Otolaryngol Head Neck Surg 2005;132:414–7.
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[9] Psaltis AJ, Ha KR, Beule AG, Tan LW, Wormald PJ. Confocal scanning laser microscopy evidence of biofilms in patients with chronic rhinosinusitis. Laryngoscope 2007;117:1302–6. [10] Prince AA, Steiger JD, Khalid AN, Dogrhamji L, Reger C, Eau Claire S, et al. Prevalence of biofilm-forming bacteria in chronic rhinosinusitis. Am J Rhinol 2008;22:239–45. [11] Rosenfeld RM, Andes D, Bhattacharyya N, Cheung D, Eisenberg S, Ganiats TG, et al. Clinical practice guideline: adult sinusitis. Otolaryngol Head Neck Surg 2007;137:S1–31. [12] Sanderson AR, Leid JG, Hunsaker DH. Bacterial biofilms on the sinus mucosa of human subjects with chronic rhinosinusitis. Laryngoscope 2006;116:1121–6. [13] Sanclement JA, Webster P, Thomas J, Ramadan HH. Bacterial biofilms in surgical specimens of patients with chronic rhinosinusitis. Laryngoscope 2005;115:578–82. [14] Foreman A, Psaltis AJ, Tan LW, Wormald PJ. Characterization of bacterial and fungal biofilms in chronic rhinosinusitis. Am J Rhinol Allergy 2009;23:556–61.
[15] Healy DY, Leid JG, Sanderson AR, Hunsaker DH. Biofilms with fungi in chronic rhinosinusitis. Otolaryngol Head Neck Surg 2008;138:641–7. [16] Foreman A, Singhal D, Psaltis AJ, Wormald PJ. Targeted imaging modality selection for bacterial biofilms in chronic rhinosinusitis. Laryngoscope 2010;120:427–31. [17] Singhal D, Psaltis AJ, Foreman A, Wormald PJ. The impact of biofilms on outcomes after endoscopic sinus surgery. Am J Rhinol Allergy 2010;24:169–74. [18] Cryer J, Schipor L, Perloff JR, Palmer JN. Evidence of bacterial biofilm in human chronic sinusitis. ORL J Otorhinolaryngol Relat Spec 2004;66:155–8. [19] Ferguson BJ, Stolz DB. Demonstration of biofilm in human bacterial chronic rhinosinusitis. Am J Rhinol 2005;19:452–7. [20] Psaltis AJ, Weitzel EK, Ha KR, Wormald PJ. The effect of bacterial biofilms and post-sinus surgical outcomes. Am J Rhinol 2008;22:1–6. [21] Zhang Z, Han D, Zhang S, Han Y, Dai W, Fan E, et al. Biofilms and mucosal healing in postsurgical patients with chronic rhinosinusitis. Am J Rhinol Allergy 2009;23:506–11.
Bacterial biofilms in chronic rhinosinusitis and their relationship with inflammation severity Hai-Hong Chen a, Xiang Liu a, Chao Ni a, Yin-Ping Lu a, Gao-Yun Xiong b, Yu-Yu Lu a, Shen-Qing Wang a,* a
Department of Head-Neck Otolaryngology, The First Affiliated Hospital, College of Medicine, Zhejiang University, Zhejiang Province, Hangzhou 310003, China b Department of Head-Neck Otolaryngology, TongDe Hospital of Zhejiang Province, Hangzhou 310013, China Received 23 December 2010; accepted 27 April 2011
Abstract Aims: To identify the presence of bacterial biofilms on mucosal specimens from chronic rhinosinusitis (CRS) patients, and evaluate their relationship with severity of CRS. Methods: A prospective study of biofilms presence on 24 CRS patients compared with 12 controls was designed. The presence of biofilms was determined by scanning electron microscopy (SEM), and associations with the preoperative Lund–MacKay CT scores, Johansson endoscopic scores, and the history of ESS were assessed. Results: Biofilms were found in 13/24 CRS patients (54.2%) but in only 1/12 controls (8.3%; P < 0.01). CRS patients with and without biofilms had similar preoperative Lund–MacKay CT and Johansson endoscopic scores (P > 0.05). Patients with revision ESS showed a tendency of higher biofilms incidence (5/7, 71.4%) than those undergoing their first procedure (8/17, 47.1%), but did not reach a significant difference (P > 0.05). Conclusions: The higher incidence of biofilms in CRS patients suggests a role in the pathogenesis of CRS, but no correlation with severity of CRS. # 2011 Elsevier Ireland Ltd. All rights reserved. Keywords: Bacterial biofilms; Chronic rhinosinusitis; Lund–MacKay CT scores; Johansson endoscopic scores; History of endoscopic sinus surgery
1. Introduction Bacterial biofilms are highly organized structures composed of bacterial communities encased within a protective extracellular matrix, which are resistant to both antibiotic treatment and host defense systems [1]. Biofilms are considered a common and important cause of persistent infections. The National Institutes of Health has estimated that at least 65% of all bacterial infections in humans are related to biofilms [2]. In the field of otolaryngology, biofilms have been documented on otitis media with effusion [3], cholesteatoma and tonsillitis [4,5], rhinosinusitis and * Corresponding author. Tel.: +86 571 87236895. E-mail address: [email protected] (S.-Q. Wang).
adenoids removed from children with chronic rhinosinusitis (CRS) [6], and they have also been isolated on some prosthetic devices, such as tracheotomy and tympanostomy tubes, front recess stents, and cochlear implants [7]. Current research suggests that biofilms may contribute greatly to the recalcitrant nature of CRS. There have been several reports regarding the presence of biofilms in surgical specimens obtained from patients with CRS. In 2005, Ramadan examined five specimens from five CRS patients, all of which were positive for biofilms [8]. Using confocal scanning laser microscopy, Psaltis found biofilms formation in 44% of 38 CRS patients [9]. A recent prospective study in over 150 consecutive CRS patients identified a positive rate of almost 30% for bacteria with biofilm-forming capacity, as assessed by an in vitro biofilm-formation assay [10].
0385-8146/$ – see front matter # 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.anl.2011.04.014
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H.-H. Chen et al. / Auris Nasus Larynx 39 (2012) 169–174
Although the association between biofilms and the recalcitrant nature of CRS has been gradually accepted by most researchers, the precise role of biofilms in the pathogenesis of CRS remains unclear. Additionally, the pathogenesis of CRS may differ between different populations and areas. There are only a few reports regarding the pathogenesis of CRS with biofilms in China, and even fewer concerning the clinical relevance of CRS with biofilms. The primary aim of this study was to determine the presence of biofilms on CRS patients in Chinese community and its correlations with Lund–Mackay CT scores, Johansson endoscopic scores, and history of endoscopic sinus surgery (ESS).
2. Materials and methods 2.1. Study design and population This was a prospective study performed to determine the presence of bacterial biofilms on the sinus mucosa of 24 CRS patients undergoing ESS compared with 12 controls without CRS. All patients were from the Otolaryngological Department of the 1st Affiliated Hospital of Zhejiang University and Zhejiang Provincial Tongde Hospital during the period July 2008 to September 2010. All subjects gave informed consent prior to enrollment in the study. All CRS patients fulfilled the diagnostic criteria for CRS according to clinical practice guidelines [11]. Clinical data, including demographic information, presence of bronchial asthma, macrolide therapy or steroid therapy before surgery, preoperative Lund–MacKay computed tomography scores, Johansson endoscopic scores and history of ESS, were recorded. Specimens were obtained intraoperatively from diseased sinuses, including the uncinate process, anterior ethmoid sinuses, posterior ethmoid sinuses or maxillary sinus, as evidenced by the preoperative CT scan. Multiple specimens were obtained per subject (47 specimens in all from 24 CRS patients), each of them varied from 3.0 mm 3.0 mm to 6.0 mm 6.0 mm. Specimens were washed thoroughly in saline, and then placed immediately into 2% glutaraldehyde for scanning electron microscopy (SEM) specimen preparation. The control group consisted of 12 patients without CRS (1 patient with nasopharyngeal carcinoma, 8 patients scheduled for nasal bone-plasty due to nasal trauma, 3 patients undergoing septoplasty). Specimens were obtained from the uncinate process or the anterior ethmoid bulla at the time of surgery.
using PBS. Next, the specimens were fixed with 1% osmium tetroxide for 1 h. They were then dehydrated through a graded ethanol series as follows: 50% for 15 min, 70% for 15 min, 80% for 15 min, 90% for 15 min, and 100% twice for 15 min each time. The tissue was immersed in 100% acetone for 15 min and washed in 100% isoamyl acetate for 15 min, followed by critical point drying. Finally, specimens were mounted on metal stubs and subsequently sputtercoated with gold preparation for imaging. Observations were carried out in our SEM laboratory using a Stereoscan 260 microscope (Leica, Wetzlar, Germany) at an acceleration voltage of 15 kV. Structures characterized by water channels, 3D structure, and matrixembedded spherical or elliptical bodies were identified as evidence of biofilms. It is differed from viscous mucous, the latter is a flat blanket, under the blanket, sometimes you may see the comparative orderly cilia, above the mucous blanket, sometimes you can find irregular foreign granule. The entire area of each specimen was scanned for the presence of biofilms structures. Images were taken at various angles to effectively display the specimens and to minimize errors and artifacts. The investigators examining the samples were blinded to the disease status of the patients. 2.3. Statistical analysis The results of this study were analyzed using the SPSS software (ver. 13.0; SPSS Inc., Chicago, IL). Continuous data were analyzed using Student’s t-test, and dichotomous data were analyzed using Fisher’s exact test. In all analyses, P values <0.05 were deemed to indicate statistical significance.
3. Results 3.1. Patient demographics and clinical data Twenty-four CRS patients and 12 controls were included in the study. The CRS group consisted of 16 males and 8 females with a mean age of 41.88 12.58 years (range 18–65 years), while the control group consisted of 8 males and 4 females with a mean age of 30.2 12.6 years (range 17–56 years). Table 1 lists the presence of bronchial asthma, macrolide therapy or steroid therapy before surgery, preoperative Lund–MacKay CT scores, Johansson endoscopic scores, and the history of ESS for each CRS patient.
2.2. Scanning electronic microscopy
3.2. SEM findings
All specimens were prepared for SEM using the following techniques. Tissue was initially fixed for 2 h in 2.5% glutaraldehyde in phosphate-buffered saline (PBS, pH 7.4) at 4 8C. Two rinses of 15 min each were then carried out
Specimens from the 24 CRS patients and 12 controls were examined for the presence of bacterial biofilms by SEM. Using strict SEM morphological criteria as well as the characteristic features of biofilms, all 24 CRS patients
H.-H. Chen et al. / Auris Nasus Larynx 39 (2012) 169–174
171
Table 1 Clinical summary and the presence of biofilms in the CRS group identified by SEM. Patients
Genderb/agea
No. No. No. No. No. No. No. No. No. No. No. No. No. No. No. No. No. No. No. No. No. No. No. No.
M/31 M/57 F/55 M/37 F/18 M/56 M/44 M/34 M/30 M/60 F/40 M/33 F/21 M/50 M/44 F/40 F/54 M/48 F/54 M/39 M/51 M/44 F/25 M/28
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
Bronchial asthma
Macrolide therapy
Steroid therapy
Previous ESS
Rhinocort (3Ms) Rhinocort (11Ms)
1 2 2 1
+(10Ys)
Rhinocort (6Ms) +(10Ys) 1 +(13Ys) +(15Ys) Rhinocort (1W)
2 1
Clarithromycin (1M)
Rhinocort (1M) Flisonase (2.5Ms)
Lund–MacKay CT scorec
Johansson endoscopic scorec
11 12 10 10 12 2 6 9 6 8 6 9 2 11 12 6 9 2 11 7 8 6 4 3
3 6 6 3 5 4 4 4 4 3 3 5 1 4 3 3 3 6 6 1 3 4 2 3
Biofilms presence + + + +
+ + + + + + + +
+
ESS: endoscopic sinus surgery; SEM: scanning electron microscopy; (+) positive; ( ) negative; n: number of previous ESS; Y: year; W: week; M: month. a In years. b M – male, F – female. c Scores of unilateral side (right or left) from which specimens taken.
showed aberrant findings on the mucosal surface with varying degrees of severity, from disarrayed cilia to the complete absence of cilia and goblet cells (Fig. 1). Characteristic morphological features of biofilms, such as water channels, 3D structure, and matrix-embedded spherical or elliptical bodies, were noted in 21 specimens from 13 cases of 24 CRS patients (54.2%, Table 1), the typical figures are showed in Fig. 2. Only 1 of the 12 controls (8.3%) showed evidence of biofilm; in the remaining 11
cases, though disarray and absence of cilia can also be found in scattered regions, the majority area of each specimen showed apparently healthy cilia and goblet cells as well as mucus blanket coating mucosa (Fig. 3), The differences in incidence rates of biofilms between CRS patients and the controls was statistically significant (Fisher’s exact test, P = 0.0079).
3.3. Correlation between the presence of bacterial biofilms and the severity of Lund–MacKay CT scores, Johansson endoscopic scores, and ESS history
Fig. 1. SEM showing denuded epithelium and scarce, disarrayed cilia in sinus mucosa of CRS patient.
Table 2 shows a comparison of the preoperative Lund– MacKay CT scores and Johansson endoscopic scores between CRS patients with and without biofilms. There was no identifiable differences in preoperative Lund– MacKay CT scores or Johansson endoscopic scores between patients with and without biofilms (Table 2). With regard to the history of ESS, among the seven CRS patients who had undergone previous ESS procedures, five were positive for biofilms. The incidence of biofilms seemed higher in patients with revision ESS (5/7, 71.4%) than in those undergoing their first procedure (8/17, 47.1%), but the difference was not statistically significant (Fisher’s exact test, P = 0.205).
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Fig. 2. SEM micrograph of biofilms in chronic rhinosinusitis patients: (A) showing 3D structure with water channels and (B) showing spherical bodies embedded in matrix.
Fig. 3. SEM appearance of a healthy control specimen showing (A) healthy epithelium and cilia and (B) a layer of mucous blanket above the cilia, with some foreign granule on the surface.
Table 2 Comparison CT and endoscopic findings between CRS patients with and without biofilm formation.
Lund–MacKay CT score Johansson endoscopic score *
Biofilm visualized (n = 13)
No biofilm visualized (n = 11)
P-value *
7.538 3.688 3.769 1.536
7.500 3.100 3.636 1.362
0.9443 0.8262
Student’s t-test.
4. Discussion Biofilms are increasingly recognized as playing an etiological role in CRS. Bacteria in biofilms are difficult to culture and largely resistant to host defense systems and antimicrobial therapy. The cultures of the planktonic bacteria in nasal discharge did not correlate with those in biofilms [12]. In fact, bacteria can be examined in normal nasal cavity, just as indicated by the study of Sanclement ‘‘All swabbed specimens, including controls, grew bacteria on routine cultures’’ [13]. In our prior study, we detected bacteria in nasal discharge in a group of 31 CRS patients, most of them were Staphylococcus epidermidis, others including Staphylococcus aureus, Pseudomonas aeruginosa, Enterobacter aerogenes, Streptococcus viridians, Staphylococcus haemo-
lyticus, Klebsiella pneumoniae, and Proteus vulgaris. But these may not quite the same as what within the biofilms, so in this study, we have not completed bacteria culture in nasal discharge. Usually the pathogens in biofilms can be exactly detected by fluorescence in situ hybridization (FISH). According to literatures, the predominant organisms were Staphylococcus aureus, Haemophilus influenzae, others including Pseudomonas aeruginosa, Streptococcus pneumophilia, and fungal elements could also be identified [12,14,15]. The incidence of biofilms in our CRS group was 54.2%, which was slightly lower than those in most previous studies. Sanclement et al. reported biofilms in 24 of 30 (80%) CRS patients using scanning electron microscopy [13]. Using confocal scanning laser microscopy (CSLM) together with
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BacLight staining or fluorescence in situ hybridization (FISH), Foreman et al. found biofilms in 18 of 20 (90%) CRS patients [16]. The reported incidence rates of biofilms in CRS patients range from 25% to 100% [9,12,13,16–19], with most studies showing rates of 70–80% [9,12,13,17]. This may be due to different populations, selection of materials for testing which represent only a small fraction of the total sinus mucosa, and also the technique used. CSLM and FISH usually have higher sensitivity and specificity than SEM, because they allow not only nondestructive analysis of the living biofilms, but also yield more detailed information on their entire structure [16]. However, regardless of the technique used, almost all previous studies demonstrated a significantly higher incidence of biofilms in CRS patients than in controls, suggesting that biofilms may play an active role in the pathogenesis of CRS. In the present study, all of the samples from the CRS group showed aberrant findings on the mucosal surface, with varying degrees of severity from disarrayed cilia to complete absence of cilia and goblet cells, even in patients without evidence of biofilms formation, while in the control group, the majority area of each specimen showed apparently normal epithelium and cilia. The mucociliary system can prevent initial bacterial approach. The disarray and absence of the mucociliary apparatus will facilitate biofilms development, and therefore reduction of sinonasal inflammation and protection of cilia represent the first step in blocking the attachment and aggregation of bacteria that would otherwise initiate biofilms formation. With regard to the correlation of biofilms presence with the severity of CRS, Singhal reported that the biofilmpositive group had significantly poorer Lund–McKay radiology and Kennedy–Lund endoscopic scores compared to those negative for biofilms, indicating more severe preoperative disease in this group of patients [17]. However, unlike the majority of reports published to date [9,17,20,21], our present study did not show a relationship between the presence of biofilms and the severities of preoperative Lund–MacKay CT and Johansson endoscopic scores between CRS patients with and without biofilms. These observations indicated that biofilms represent just one state in which bacteria exist, and biofilm-associated bacteria may have stronger resistance to both antibiotic treatment and host defense, resulting in recalcitrant infection. However, biofilms may not parallel the severity of inflammation, because the severity of CRS inflammation is multifactorial. It should be noted that the lack of correlation in the present study may have been due to the differences in study methods used, the patient population, and random specimen selection as well as subjective evaluation. Additionally, the cohort in the present study was small, and further studies with larger sample sizes are required. Although there was no statistically significant relationship between the presence of biofilms and a history of previous sinus surgery, patients with revision ESS tended to show a higher rate of biofilms than those undergoing their
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primary surgery, which in agreement with findings reported previously by Psaltis and Singhal [9,17]. Due to the small number of cases with a history of previous sinus surgery in the present study, further studies with larger sample sizes are needed. Prince reported a significant correlation between the presence of biofilms and the number of prior ESS procedures [10], which suggested that biofilms may contribute to the recurrence of CRS and result in unfavorable outcomes of ESS as residual biofilms left behind after surgery may serve as a nidus for bacterial microcolony and biofilm regeneration, and thus act as stimuli for an ongoing inflammatory response. Although the presence of biofilms in CRS has been suggested previously, the present study added further evidence of the existence of biofilms in the Chinese community. The presence of biofilms may partially explain the recurrent nature of CRS and its refractoriness to surgical intervention. Further studies with larger sample sizes are required to assess the relationship between the presence of biofilms and the severity of CRS, as well as risk factors contributing to biofilms formation.
Conflict of interest None.
Acknowledgement Project Foundation: The Project Supported by Medical Scientific Research Foundation of Zhejiang Province, China (no. 2008B083).
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