Inhibitory Effect of Thymol on Tympanostomy Tube Biofilms of Methicillin-Resistant Staphylococcus aureus and Ciprofloxacin-Resistant Pseudomonas aeruginosa

Abstract

The formation of antibiotic-resistant strain biofilms in tympanostomy tubes results in persistent and refractory otorrhea. In the present study, we investigated the in vitro antibiofilm activity of thymol against biofilms formed by methicillin-resistant Staphylococcus aureus (MRSA) and ciprofloxacin-resistant Pseudomonas aeruginosa (CRPA), using live and dead bacterial staining and adhesion, biofilm formation, biofilm eradication, and biofilm hydrolytic activity assays. The antibiofilm activity of thymol against tympanostomy tube biofilms formed by MRSA and CRPA strains was examined using a scanning electron microscope. In response to thymol treatment, we detected significant concentration-dependent reductions in the viability and adhesion of MRSA and CRPA. Exposure to thymol also inhibited the formation of both MRSA and CRPA biofilms. Furthermore, thymol was observed to enhance the eradication of preformed mature biofilms produced by MRSA and CRPA and also promoted a reduction in the rates of MRSA and CRPA hydrolysis. Exposure to thymol eradicated extracellular polysaccharide present in the biofilm matrix produced by MRSA and CRPA. Additionally, thymol was observed to significantly eradicate MRSA and CRPA biofilms that had formed on the surface on tympanostomy tubes. Collectively, our findings indicate that thymol is an effective inhibitor of MRSA and CRPA biofilms, and accordingly has potential utility as a therapeutic agent for the treatment of biofilm-associated refractory post-tympanostomy tube otorrhea resulting from MRSA and CRPA infection.

Keywords: biofilm; ciprofloxacin-resistant Pseudomonas aeruginosa; methicillin-resistant Staphylococcus aureus; thymol; tympanostomy tube.

Figure 1

The effects of thymol on the viability of methicillin-resistant Staphylococcus aureus (MRSA) and ciprofloxacin-resistant Pseudomonas aeruginosa (CRPA). MRSA and CRPA viabilities were examined following 24 h exposure to different concentrations of thymol. Thymol promoted significant concentration-dependent reductions in live MRSA and CRPA cells (green), and increases in dead MRSA and CRPA cells (red). Data are shown as the means ± standard errors of the mean of five independent experiments (** p < 0.01; * p < 0.05).

Figure 2

The effects of thymol on the adhesion of methicillin-resistant Staphylococcus aureus (MRSA) and ciprofloxacin-resistant Pseudomonas aeruginosa (CRPA). The adherent MRSA and CRPA strains were stained using crystal violet following a 30 min treatment with thymol (top). Thymol significantly reduced the adhesion of MRSA and CRPA in a concentration-dependent manner. Data are shown as the means ± standard errors of the mean of five independent experiments (** p < 0.01; * p < 0.05).

Figure 3

The activity of thymol against the formation of biofilms produced by methicillin-resistant Staphylococcus aureus (MRSA) and ciprofloxacin-resistant Pseudomonas aeruginosa (CRPA). Biofilm formation was examined following 24 h exposure to thymol. Thymol significantly inhibited the rates of MRSA and CRPA biofilm formation in a concentration-dependent manner. Data are shown as the means ± standard errors of the mean of five independent experiments (** p < 0.01, * p < 0.05).

Figure 4

The activity of thymol against the preformed biofilms of methicillin-resistant Staphylococcus aureus (MRSA) and ciprofloxacin-resistant Pseudomonas aeruginosa (CRPA). Eradication and hydrolytic activity were determined following treatment with thymol for 24 h. The rates of MRSA and CRPA biofilm eradication increased in response to exposure to increasing concentrations of thymol. The minimum biofilm eradication concentrations (MBECs) of thymol by optical density measurements against preformed MRSA and CRPA biofilms were 0.78 and 3.13 mg/mL, respectively (A). The MBEC of thymol by viable cell counts against preformed MRSA and CRPA biofilms were 0.78 and 6.25 mg/mL, respectively (B). The hydrolytic activity of biofilms produced by MRSA and CRPA decreased significantly in response to exposure to increasing concentrations of thymol (C). Data are shown as the means ± standard errors of the mean of five independent experiments (CFU; colony-forming units, FDA; fluorescein diacetate, ** p < 0.01; * p < 0.05).

Figure 5

The activity of thymol against the extracellular matrix of biofilms produced by methicillin-resistant Staphylococcus aureus (MRSA) and ciprofloxacin-resistant Pseudomonas aeruginosa (CRPA). The extracellular matrix was determined by staining with WGA-Alexa 488 following treatment with thymol for 24 h. The extracellular matrix of biofilms produced by MRSA and CRPA decreased significantly in response to exposure to increasing concentrations of thymol. Data are shown as the means ± standard errors of the mean of five independent experiments (WGA; wheat germ agglutinin, DAPI; 4′,6-diamidino-2-phenylindole, ** p < 0.01; * p < 0.05).

Figure 6

Scanning electron micrographs of methicillin-resistant Staphylococcus aureus (MRSA) and ciprofloxacin-resistant Pseudomonas aeruginosa (CRPA) biofilms on the surface of tympanostomy tubes after 24 h exposure to thymol. Thymol was found to concentration-dependently reduce the MRSA and CRPA biofilms and colonies.