The Therapeutic Effect of 1,8-Cineol on Pathogenic Bacteria Species Present in Chronic Rhinosinusitis

Abstract

Chronic rhinosinusitis (CRS) is marked by an inflamed mucosa of sinuses and is accompanied by a significantly reduced quality of live. Since no guidelines for the treatment of CRS are available, long lasting clinical histories with health care costs adding up to dozens of billion $ annually are caused by CRS. The progression of CRS is often induced by bacterial infections and/or a shift in microbiome as well as biofilm formation. The exact microbiome alterations are still unclear and the impenetrable biofilm renders the treatment with common antibiotics ineffective. This study focuses on characterizing the microbiome changes in CRS and investigating the inhibition of biofilm growth by 1,8-Cineol, a small, non-polar and hence biofilm penetrating molecule with known antimicrobial potential. We performed MALDI-TOF MS based characterization of the microbiomes of healthy individuals and CRS patients (n = 50). The microbiome in our test group was shifted to pathogens (Staphylococcus aureus, Escherichia coli, and Moraxella catarrhalis). In contrast to published studies, solely based on cell culture techniques, we could not verify the abundance of Pseudomonas aeruginosa in CRS. The inhibition of bacterial proliferation and biofilm growth by 1,8-Cineol was measured for these three pathogens. Interestingly, S. aureus, the most prominent germ in CRS, showed a biofilm inhibition not simply correlated to its inhibition of proliferation. RT-qPCR confirmed that this was due to the downregulations of major key players in biofilm generation (agrA, SarA and σ^B) by 1,8-Cineol. Furthermore we verified this high biofilm inhibition potential in a model host system consisting out of S. aureus biofilm grown on mature respiratory epithelium. A second host model, comprising organotypic slices, was utilized to investigate the reaction of the innate immune system present in the nasal mucosa upon biofilm formation and treatment with 1,8-Cineol. Interestingly Staphylococcus epidermidis, the cause of very common catheter infections, possesses a biofilm generation pathway very similar to S. aureus and might be treatable in a similar fashion. The two presented in vitro model systems might be transferred to combinations of every biofilm forming bacterial with most kind of epithelium and mucosa.

Keywords: 1; 8-Cineol; S. aureus; biofilm; chronic rhinosinusitis; host model system.

FIGURE 1

The normal microbiome compared to the microbiome of CRS patients. (A) MALDI-TOF-MS-based analysis of the microbiome of CRS patients and a healthy control group with clinical standardized pre-culture (n = 50). The commensal Corynebacterium and viridans Streptococci species are more frequent in healthy sinuses, while potential pathogenic bacteria like E. coli and M. catarrhalis are more often detectable in CRS patients. (B) MALDI-TOF-MS-based analysis of the microbiomes with pre-cultivation on Baird-Parker agar. The commensal bacterium S. epidermidis occurs less frequent in CRS patients at the same time the potential pathogen S. aureus can be detected more often (^∗≤0.05; binomial test; one-tailed).

FIGURE 2

The antibacterial properties of 1,8-Cineol on different CRS related pathogens. (A) The MIC was determined for E. coli, M. catarrhalis and S. aureus after 6 h of incubation with the indicated concentration of 1,8-Cineol. The bacteria M. catarrhalis was the least susceptible exhibiting a MIC of 5 mg/ml also S. aureus showed a slight growth at 2.5 mg/ml only the growth of E. coli was completely inhibited by a concentration of 2.5 mg/ml 1,8-Cineol. (B) The MBIC₅₀ was measured for the pure culture of all three different bacterial strains. The germs M. catarrhalis and E. coli showed the smallest inhibition of biofilm growth upon exposure to 1,8-Cineol with MBIC₅₀ values between 1.25 mg/ml and 0.625 mg/ml and around 0.625 mg/ml, respectively. S. aureus showed a high sensitivity in terms of biofilm growth with a MBIC₅₀ of only 0.15 mg/ml. (C) RT-qPCR analysis of the transcripts of the major key players in biofilm generation of S. aureus upon treatment with 1 mg/ml 1,8-Cineol normalized to the initial expression level of the inoculum. All three key players are downregulated upon treatment with 1,8-Cineol. After 2 h only subtle differences are detectable; after 6 h of incubation the expression level in sessile and planktonic bacteria treated with 1,8-Cineol is already significantly reduced. After 24h of biofilm maturation this difference becomes much more prominent and the corresponding expression levels of 1,8-Cineol treated sessile bacteria make up only a fraction of the non-treated ones (^∗∗ ≤ 0.01, ^∗ ≤ 0.05, ns > 0.05; unpaired t-test; one-tailed, confidence interval: 95%).

FIGURE 3

Generation of a S. aureus biofilm and its effect on the in vitro host models of CRS. (A) Determination of the MBIC₅₀ in an in vitro host model via a MTT assay. The metabolism of the co-culture is the sum of the metabolism of the respiratory epithelium and the biofilm formed by S. aureus (left). When the metabolism of the underlying epithelium (epi.) is subtracted from the metabolism of the co-culture (right), a MBIC₅₀ between 0,125 and 0.0625 mg/ml 1,8-Cineol can be derived. The amount of biofilm grown without underlying epithelium (no epi.) is about half as much as with epithelium, emphasizing the biofilm promoting properties of the epithelium. (B) A RT-qPCR based approach to measure the MBIC₅₀ in the host model. After extraction of gDNA from the viable bacterial cells the measured amount of gDNA lead to an MBIC₅₀ around 0.125 mg/ml 1,8-Cineol. (C) 3D-reconstruction of confocal laser scanning microscopic data (left) and a cross section (right) showing an organotypic slice co-cultivated with S. aureus. The biofilm is solely stained with DAPI (blue) while the eukaryotic actin filaments are also stained with Phalloidin (magenta). The biofilm comprises a thickness between 5 and 15 μm and is separated from the underlying respiratory epithelium by a mucus layer spanning over 10–30 μm. (D) Scanning electron microscopic image of a biofilm grown on organotypic slices. The biofilm homogenously covers the respiratory mucosa and consist out of a dense network of EPS embedding the bacterial cells. High resolution images show that some cells secrete these EPS (arrowheads) while other cells are dividing (arrows). (E) Comparison of cytokine transcription of the uninfected host model (C) to the biofilm infected model (BF) and the reaction upon treatment with 1,8-Cineol (BF + Cineol): IL 6 and GM-CSF were upregulated by the biofilm infection. The treatment with 1,8-Cineol dampened this inflammatory reaction markedly. (F) RT-qPCR investigation of target genes of the NF-kB pathway A20 and IKB-α. Both transcripts are upregulated by the biofilm growth. The addition of 1,8-Cineol results in a further upregulation of these two negative regulators. (G) RT-qPCR data of the relative expression of antimicrobial peptides showing a slight upregulation upon biofilm growth and a very strong downregulation upon treatment with 1,8-Cineol (^∗∗∗ ≤ 0.001, ^∗∗ ≤ 0.01, ^∗ ≤ 0.05; unpaired t-test; one-tailed, confidence interval: 95%).