Evaluation of Zoono® on biofilms in chronic rhinosinusitis

Introduction: Chronic rhinosinusitis (CRS) is a condition involving persistent inflammation of the sinonasal mucosa, corresponding to a poor quality of life. There are many contributors to the pathogenesis of CRS, including biofilms, which are a group of microbes embedded in an extracellular matrix. However, the detection of biofilms in the sinuses of CRS patients has largely been limited to culture-based and microscopy methods. Significantly, there are no treatment options for biofilm-positive CRS. This necessitates the search for alternative antimicrobial agents that target biofilms. Zoono, a commerciallyavailable antiseptic, has shown promise at eradicating biofilms of Staphylococcus aureus and Pseudomonas aeruginosa. However, the antibiofilm activity against other clinically-relevant bacteria commonly found in the sinuses has not been elucidated. Materials and Methods: A wide range of reference s:pecies and clinical strains were optimised for growth on the Calgary Biofilm Device (CBD) due to their relevance in CRS and other conditions. These species include Staphylococcus aureus, Pseudomonas aeruginosa, Staphylococcus epidermidis, Moraxella catarrhalis, Streptococcus pneumoniae, Streptococcus pyogenes, Corynebacterium accolens, Dolosigranulum pigrum, Klebsiella pneumoniae, Acinetobacter baumannii, and Escherichia coli. Here, trialling of different conditions, such as shaking speed, supplementation, and the presence of CO2 occurred. The extent of biofilm growth was measured using mean log Colony Forming Units (CFU)/peg and then compared among the different conditions. Once growth conditions were established, time series experiments were performed to determine the susceptibility of the biofilms to antibiofilm agents. This included four reference strains K. pneumoniae, Methicillin-resistant S. aureus, A. baumannii, E. coli, and eight clinical strains of S. aureus and P. aeruginosa with different antibiotic susceptibilities. Here, each strain and species were tested against Zoono and the modified formulation, Zoono B23-1402A, for different exposure times of 5 min, 30 min, and 6 h. The planktonic cells and biofilm cells, either untreated or treated with doxycycline (0.27 µg/mL) or Zoono, of eight reference strains of species found in the sinuses, were grown in 6-well plates and then collected for RNA extraction. This included methicillin-sensitive and -resistant S. aureus, P. aeruginosa, S. epidermidis, C. accolens, D. pigrum, S. pneumoniae, and M. catarrhalis. RNA from each sample underwent RNA sequencing and was processed bioinformatically for intra- and interspecies analyses. Results: Most species used in this study showed evidence of biofilm growth on the CBD except for S. pneumoniae ATCC 49619, D. pigrum ATCC 51524, and multiple reference strains of S. pyogenes. Zoono and Zoono B23- 1402A showed biofilm eradication for all species and strains after exposure of 6 h. However, there was species- and strain-dependent variation for the extent of biofilm killing between 5 min and 30 min exposure times. Zoono B23-1402A generally achieved biofilm eradication faster than Zoono. Intraspecies gene expression analysis showed a general upregulation in quorum sensing processes and downregulation of cell growth and metabolism processes for untreated biofilm cells compared to planktonic cells. Doxycycline-treated biofilm cells showed an upregulation of cell metabolism and growth processes compared to untreated biofilm cells. Zoono-treated biofilm cells had more variable regulations in processes, with some species showing upregulation in cell death or cell growth. Only 207 genes were found to be common across planktonic and biofilm (untreated) samples of the eight species. However, among these common genes, no specific gene(s) could be identified to be associated uniquely with biofilms. Conclusion: The CBD was a versatile platform for growing biofilms and testing antibiofilm agents. Zoono and especially Zoono B23-1402A were effective antibiofilm agents against a wide range of bacterial species and strains. There was high variation in the transcriptome profiles of bacterial species found in the sinuses depending on the cell state and exposure to treatment. There may not be a gene found among species that are differentially expressed in biofilms. However, a more in-depth analysis would provide further information regarding biofilm-specific genes found across all eight species, antimicrobial resistance genes in the biofilms before and after treatment, and finally, the mechanisms of action of the antiseptic chosen in this study.

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