## 5 Discussion  
Even though the minimal inhibitory concentration (MIC) of vancomycin is sufficient in the serum, the MIC may be insufficient for reducing bacterial proliferation in the infected area. Particularly in the infected parietal pleura, the mesothelial cells that line the pleural cavity, which is an avascular area, may already be thickened, and the MIC of vancomycin may not be enough to allow sufficient vancomycin to penetrate the thickened pleura. Vancomycin is considered as \"time-dependent antibiotics\", which is not dependent on the \"concentration\"; therefore, a larger area under the curve (AUC) correlated with a superior effect [4; 5; 6].  
Since there are few reports describing the pharmacokinetics of vancomycin in the pleural cavity, we measured the vancomycin serum and pleural levels in in pleural cavity after injection via pleura and blood. Our findings revealed that the concentrations of the serum and pleural fluid in 6 h after installation in pleura were at a therapeutic level. Although the mechanism of resorption of vancomycin through the pleura is unclear, the prolonged AUC of vancomycin in the pleural cavity ensured eradication of the infection without injection of additional vancomycin in the serum.  
The mesothelial cells that line the pleural cavity have an important role in the filtration of the pleural fluid, resulting from the difference in the hydrostatic and colloid-osmotic pressure between the pleural fluid and capillary blood. Furthermore, it has been predicted that mesothelial cells have the ability to reabsorb antibiotics such as vancomycin, which are applied as topical agents in the thoracic cavity [7]. However, this has not been demonstrated in animal experiments.  
After vancomycin injection in the pleural cavity of a rat, the phenomenon that the peak concentration in the thickened pleura reached 15 min earlier than the one in the serum might occur because the inflammatory stimulation alters the endothelial barrier function to allow the high permeability of antibiotics in the pleura (Figure 2C,D).  
Although the injected vancomycin easily penetrated the thickened pleura into the serum in the early period, the concentration of vancomycin could be maintained above the trough level in the pleural cavity with the prolonged AUC of vancomycin, which suggest that an effective topical concentration could be maintained even in thickened pleura [5]. To maintain over the trough level in pleural cavity, intrapleural installation of vancomycin needs to be repeated every four hours.  
Comparing with the cases injected into the vessel, the concentration of injected vancomycin in pleura barely achieved the trough level during first one hour (Figure 2E). This phenomenon might be explained by the fact that in the thickened parietal pleura, which is an avascular area, the MIC of vancomycin may not be enough to allow sufficient vancomycin to penetrate the thickened pleura. This experiment provides evidence that irrigation of the pleural cavity with an antibiotic solution containing vancomycin may be an efficient treatment strategy, especially in the case of an MRSA infection in the thickened pleura. Owing to the difficulty in creating an animal model of chronic empyema, the efficacy of topically applied antibiotics in a thickened pleural cavity has not been sufficiently studied. In future, research should evaluate the pharmacokinetics of topically applied antibiotics in the pleural cavity of animals.  
Conceptualisation, S.L. and H.Y.A.; methodology, K.K.; formal analysis, S.Y.M.; data curation, J.H.K.; writing--original draft preparation, S.L. and H.Y.A.; supervision, H.Y.A.; and project administration, Y.D.K. H.Y.A. and K.K. contributed equally to this study. All authors have read and agreed to the published version of the manuscript.  
This research received no external funding.  
All procedures were approved by the Pusan National University Hospital's Institutional Animal Care and Use Committee (No. 2020-165).  
Not applicable.  
The authors declare no conflict of interest.