The microbiome of chronic rhinosinusitis: culture, molecular diagnostics and biofilm detection

Sam Boase, Andrew Foreman, Edward Cleland, Lorwai Tan, Rachel Melton-Kreft, Harshita Pant, Fen Z Hu, Garth D Ehrlich, Peter-John Wormald, Sam Boase, Andrew Foreman, Edward Cleland, Lorwai Tan, Rachel Melton-Kreft, Harshita Pant, Fen Z Hu, Garth D Ehrlich, Peter-John Wormald

Abstract

Background: Bacteria and fungi are believed to influence mucosal inflammation in chronic rhinosinusitis (CRS). However their presence and relationship to disease is debated. This study used multiple detection methods to compare microbial diversity and microbial abundance in healthy and diseased sinonasal mucosa. The utility of contemporary detection methods is also examined.

Methods: Sinonasal mucosa was analyzed from 38 CRS and 6 controls. Bacterial and fungal analysis was performed using conventional culture, molecular diagnostics (polymerase chain reaction coupled with electrospray ionization time-of-flight mass spectrometry) and fluorescence in situ hybridization.

Results: Microbes were detected in all samples, including controls, and were often polymicrobial. 33 different bacterial species were detected in CRS, 5 in control patients, with frequent recovery of anaerobes. Staphylococcus aureus and Propionibacterium acnes were the most common organisms in CRS and controls, respectively. Using a model organism, FISH had a sensitivity of 78%, and a specificity of 93%. Many species were detected in both CRS and controls however, microbial abundance was associated with disease manifestation.

Conclusions: This study highlights some cornerstones of microbial variations in healthy and diseased paranasal sinuses. Whilst the healthy sinus is clearly not sterile, it appears prevalence and abundance of organisms is critical in determining disease. Evidence from high-sensitivity techniques, limits the role of fungi in CRS to a small group of patients. Comparison with molecular analysis suggests that the detection threshold of FISH and culture is related to organism abundance and, furthermore, culture tends to select for rapidly growing organisms.

Figures

Figure 1
Figure 1
Staphylococcus aureus FISH probe tagged with Alexa 488, analyzed with confocal scanning laser microscopy at 80× magnification.
Figure 2
Figure 2
The relationship between S. aureus abundance, and detection by FISH and culture. The detection of S. aureus by FISH and culture is dependant on the abundance of the bacteria as measured by the number of bacterial genomes present in the sample.

References

    1. Amann RI, Ludwig W, Schleifer KH. Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol Rev. 1995;59:143–169.
    1. Costerton JW, Stewart PS, Greenberg EP. Bacterial biofilms: A common cause of persistent infections. Science. 1999;284:1318–1322. doi: 10.1126/science.284.5418.1318.
    1. Singhal D, Psaltis AJ, Foreman A, Wormald PJ. The impact of biofilms on outcomes after endoscopic sinus surgery. Am J Rhinol Allergy. 2010;24:169–174. doi: 10.2500/ajra.2010.24.3462.
    1. Foreman A, Holtappels G, Psaltis AJ. Adaptive immune responses in Staphylococcus aureus biofilm-associated chronic rhinosinusitis. Allergy: European Journal of Allergy and Clinical Immunology. 2011;66:1449–1456. doi: 10.1111/j.1398-9995.2011.02678.x.
    1. Davies CE, Hill KE, Wilson MJ. Use of 16S ribosomal DNA PCR and denaturing gradient gel electrophoresis for analysis of the microfloras of healing and nonhealing chronic venous leg ulcers. J Clin Microbiol. 2004;42:3549–3557. doi: 10.1128/JCM.42.8.3549-3557.2004.
    1. Foreman A, Singhal D, Psaltis AJ, Wormald PJ. Targeted imaging modality selection for bacterial biofilms in chronic rhinosinusitis. Laryngoscope. 2009;120:427–431.
    1. Borriello G, Werner E, Roe F, Kim AM, Ehrlich GD, Stewart PS. Oxygen limitation contributes to antibiotic tolerance of Pseudomonas aeruginosa in biofilms. Antimicrob Agents Chemother. 2004;48:2659–2664. doi: 10.1128/AAC.48.7.2659-2664.2004.
    1. Costerton JW, Post JC, Ehrlich GD. New methods for the detection of orthopedic and other biofilm infections. FEMS Immunol Med Microbiol. 2010;61:133–140.
    1. Dunbar J, White S, Forney L. Genetic diversity through the looking glass: Effect of enrichment bias. Appl Environ Microbiol. 1997;63:1326–1331.
    1. Benninger MS, Ferguson BJ, Hadley JA. Adult chronic rhinosinusitis: definitions, diagnosis, epidemiology, and pathophysiology. Otolaryngol Head Neck Surg. 2003;129:S1–S32. doi: 10.1016/S0194-5998(03)01397-4.
    1. Foreman A, Psaltis AJ, Tan LW, Wormald PJ. Characterization of bacterial and fungal biofilms in chronic rhinosinusitis. Am J Rhinol Allergy. 2009;23:556–561. doi: 10.2500/ajra.2009.23.3413.
    1. Jiang Y, Hofstadler SA. A highly efficient and automated method of purifying and desalting PCR products for analysis by electrospray ionization mass spectrometry. Anal Biochem. 2003;316:50–57. doi: 10.1016/S0003-2697(03)00024-1.
    1. Ecker DJ, Sampath R, Massire C. Ibis T5000: A universal biosensor approach for microbiology. Nat Rev Microbiol. 2008;6:553–558. doi: 10.1038/nrmicro1918.
    1. Singhal D, Foreman A, Bardy JJ, Wormald PJ. Staphylococcus aureus biofilms: Nemesis of endoscopic sinus surgery. Laryngoscope. 2011;121:1578–1583. doi: 10.1002/lary.21805.
    1. Jervis-Bardy J, Foreman A, Field J, Wormald PJ. Impaired mucosal healing and infection associated with Staphylococcus aureus after endoscopic sinus surgery. Am J Rhinol Allergy. 2009;23:549–552. doi: 10.2500/ajra.2009.23.3366.
    1. Foreman A, Wormald PJ. Different biofilms, different disease? A clinical outcomes study. Laryngoscope. 2010;120:1701–1706. doi: 10.1002/lary.21024.
    1. Stephenson MF, Mfuna L, Dowd SE. Molecular characterization of the polymicrobial flora in chronic rhinosinusitis. J Otolaryngol Head Neck Surg. 2010;39:182–187.
    1. Healy DY, Leid JG, Sanderson AR, Hunsaker DH. Biofilms with fungi in chronic rhinosinusitis. Otolaryngol Head Neck Surg. 2008;138:641–647. doi: 10.1016/j.otohns.2008.02.002.
    1. Sanderson AR, Leid JG, Hunsaker D. Bacterial biofilms on the sinus mucosa of human subjects with chronic rhinosinusitis. Laryngoscope. 2006;116:1121–1126. doi: 10.1097/01.mlg.0000221954.05467.54.
    1. Stressmann FA, Rogers GB, Chan SW. Characterization of bacterial community diversity in chronic rhinosinusitis infections using novel culture-independent techniques. Am J Rhinol Allergy. 2011;25:e133–e140. doi: 10.2500/ajra.2011.25.3628.
    1. Hamad WA, Matar N, Elias M. Bacterial flora in normal adult maxillary sinuses. Am J Rhinol Allergy. 2009;23:261–263. doi: 10.2500/ajra.2009.23.3317.
    1. Larson DA, Han JK. Microbiology of sinusitis: Does allergy or endoscopic sinus surgery affect the microbiologic flora? Curr Opin Otolaryngol Head Neck Surg. 2011;19:199–203. doi: 10.1097/MOO.0b013e328344f67a.
    1. Psaltis AJ, Ha KR, Beule AG, Tan LW, Wormald PJ. Confocal scanning laser microscopy evidence of biofilms in patients with chronic rhinosinusitis. Laryngoscope. 2007;117:1302–1306. doi: 10.1097/MLG.0b013e31806009b0.
    1. Galli J, Calo L, Ardito F. Damage to ciliated epithelium in chronic rhinosinusitis: What is the role of bacterial biofilms. Ann Otol Rhinol Laryngol. 2008;117:902–908.
    1. Pereira F, Carneiro J, Amorim A. Identification of species with DNA-based technology: Current progress and challenges. Recent Pat DNA Gene Seq. 2008;2:187–200. doi: 10.2174/187221508786241738.
    1. Ebbens FA, Georgalas C, Fokkens WJ. Fungus as the cause of chronic rhinosinusitis: The case remains unproven. Curr Opin Otolaryngol Head Neck Surg. 2009;17:43–49. doi: 10.1097/MOO.0b013e32831de91e.
    1. Lackner A, Stammberger H, Buzina W. Fungi: A normal content of human nasal mucus. Am J Rhinol. 2005;19:125–129.
    1. Catten MD, Murr AH, Goldstein JA, Mhatre AN, Lalwani AK. Detection of fungi in the nasal mucosa using polymerase chain reaction. Laryngoscope. 2001;111:399–403. doi: 10.1097/00005537-200103000-00006.
    1. Ponikau J, Sherris D, Kern E. The diagnosis and incidence of allergic fungal sinusitis. Mayo Clin Proc. 1999;74:877–884. doi: 10.4065/74.9.877.
    1. Rao AK, Mathers PH, Ramadan HH. Detection of fungi in the sinus mucosa using polymerase chain reaction. Otolaryngol Head Neck Surg. 2006;134:581–585. doi: 10.1016/j.otohns.2005.10.047.
    1. Eyigor H, Eyigor M, Gunel C, Gultekin B, Basak S, Aydin N. Characterization of fungi in chronic rhinosinusitis using polymerase chain reaction and sequencing. Eur Arch Otorhinolaryngol. 2008;265:651–655. doi: 10.1007/s00405-007-0529-0.
    1. McCann KS. The diversity-stability. Nature. 2000;405:228–233. doi: 10.1038/35012234.
    1. Gontcharova V, Youn E, Sun Y, Wolcott RD, Dowd SE. A comparison of bacterial composition in diabetic ulcers and contralateral intact skin. Open Microbiol J. 2010;4:8–19. doi: 10.2174/1874285801004010008.
    1. Hansen J, Gulati A, Sartor RB. The role of mucosal immunity and host genetics in defining intestinal commensal bacteria. Curr Opin Gastroenterol. 2010;26:564–571. doi: 10.1097/MOG.0b013e32833f1195.
    1. Round JL, Mazmanian SK, Round JL, Mazmanian SK. The gut microbiota shapes intestinal immune responses during health and disease. Nat Rev Immunol. 2009;9:313–323. doi: 10.1038/nri2515.
    1. Macpherson AJ, Harris NL, Macpherson AJ, Harris NL. Interactions between commensal intestinal bacteria and the immune system. Nat Rev Immunol. 2004;4:478–485. doi: 10.1038/nri1373.
    1. Sekirov I, Russell SL, Antunes LC. Gut microbiota in health and disease. Physiol Rev. 2010;90:859–904. doi: 10.1152/physrev.00045.2009.
    1. Fujimura S, Nakamura T. Purification and properties of a bacteriocin-like substance (Acnecin) of oral Propionibacterium acnes. Antimicrob Agents Chemother. 1978;14:893–898. doi: 10.1128/AAC.14.6.893.
    1. Lysenko ES, Lijek RS, Brown SP, Weiser JN. Within-host competition drives selection for the capsule virulence determinant of streptococcus pneumoniae. Curr Biol. 2010;20:1222–1226. doi: 10.1016/j.cub.2010.05.051.
    1. Sibley CD, Duan K, Fischer C. Discerning the complexity of community interactions using a Drosophila model of polymicrobial infections. PLoS Pathog. 2008;4:art. no. e1000184.

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