A Novel Approach to Study the Effect of Ciprofloxacin on Biofilms of Corynebacterium spp. Using Confocal Laser Scanning Microscopy.

Non-diphtherial corynebacteria are Gram-positive rods that cause opportunistic infections, what is supported by their ability to produce biofilm on artificial surfaces. In this study, the characteristic of the biofilm produced on vascular and urological catheters was determined using a confocal microscopy for the most frequently involved in infections diphtheroid species. They were represented by the reference strains of Corynebacterium striatum ATCC 6940 and C. amycolatum ATCC 700207. The effect of ciprofloxacin on the biofilm produced by the antibiotic-susceptible C. striatum strain was evaluated using three concentrations of the antimicrobial agent (2 ×, 4 ×, and 6 × the MIC - the Minimum Inhibitory Concentration). The basis for the interpretation of results was the statistical analysis of maximum points readings from the surface comprising a total of 245 areas of the biofilm image under the confocal microscope. It was observed that ciprofloxacin at a concentration equal to 4 × MIC paradoxically caused an enlargement of areas with live bacteria within the biofilm. Biofilm destruction required the application of ciprofloxacin at a concentration higher than 6 × MIC. This suggests that the use of relatively low doses of antimicrobial agents may increase the number of live bacteria within the biofilm, and further facilitate their detachment from the biofilm's structure thus leading to the spread of bacteria into the bloodstream or to the neighboring tissues. The method of biofilm analysis presented here provides the original and novel approach to the investigation of the diphtheroid biofilms and their interaction with antimicrobial agents. Non-diphtherial corynebacteria are Gram-positive rods that cause opportunistic infections, what is supported by their ability to produce biofilm on artificial surfaces. In this study, the characteristic of the biofilm produced on vascular and urological catheters was determined using a confocal microscopy for the most frequently involved in infections diphtheroid species. They were represented by the reference strains of Corynebacterium striatum ATCC 6940 and C. amycolatum ATCC 700207. The effect of ciprofloxacin on the biofilm produced by the antibiotic-susceptible C. striatum strain was evaluated using three concentrations of the antimicrobial agent (2 ×, 4 ×, and 6 × the MIC – the Minimum Inhibitory Concentration). The basis for the interpretation of results was the statistical analysis of maximum points readings from the surface comprising a total of 245 areas of the biofilm image under the confocal microscope. It was observed that ciprofloxacin at a concentration equal to 4 × MIC paradoxically caused an enlargement of areas with live bacteria within the biofilm. Biofilm destruction required the application of ciprofloxacin at a concentration higher than 6 × MIC. This suggests that the use of relatively low doses of antimicrobial agents may increase the number of live bacteria within the biofilm, and further facilitate their detachment from the biofilm’s structure thus leading to the spread of bacteria into the bloodstream or to the neighboring tissues. The method of biofilm analysis presented here provides the original and novel approach to the investigation of the diphtheroid biofilms and their interaction with antimicrobial agents.