Is transmission electron microscopy (TEM) a promising approach for qualitative and quantitative investigations of polymyxin B and miconazole interactions with cellular and subcellular structures of Staphylococcus pseudintermedius, Escherichia coli, Pseudomonas aeruginosa and Malassezia pachydermatis?

Antimicrobial therapy using a combination of polymyxin B and miconazole is effective against the main bacterial pathogens associated with otitis externa in dogs, and a synergistic effect of both drugs has been shown previously. The objective of the present investigation was to visualize ultrastructural changes after exposure of Escherichia coli, Pseudomonas aeruginosa, Staphylococcus pseudintermedius and Malassezia pachydermatis to polymyxin B and miconazole by transmission electron microscopic (TEM). For this, cultures of E. coli, P. aeruginosa, S. pseudintermedius and M. pachydermatis were exposed to polymyxin B and miconazole, alone or in combination for 24 h. Ultrastructural changes were observed most frequently in the cell envelope of the four microorganisms. Exposure to polymyxin B seemed to cause more damage than miconazole within the range of concentrations applied. Treatment resulted in changes of the cell size: in E. coli, cell size increased significantly after treatment with either compound alone; in P. aeruginosa, cell size decreased significantly after treatment with polymyxin B and with miconazole; exposure of S. pseudintermedius to miconazole caused a decrease in cell size; in M. pachydermatis, cell size increased significantly after treatment with polymyxin B.; in E.coli, S. pseudintermedius and M. pachydermatis, cell size changed highly significant, in P. aeruginosa significantly after exposure to the combination of both compounds. In conclusion, by using a different approach than previous investigations, this study confirmed a clear combinatory effect of polymyxin B and miconazole against the tested microorganisms involved in canine otitis externa. It is the first time that visualization technologies were applied to compare the effect of single drugs to their combinatory effects on cellular and subcellular entities of selected bacterial and yeast species.