*3.3. Determination of Susceptibility Profiles of Bacterial Biofilms*

It is well established that biofilms have greater resistance to antimicrobials than planktonic cells [26]. There are several explanations for this increase in resistance. The extracellular matrix surrounding the cells in the biofilm prevents targeting and subsequent penetration by antimicrobials. The reduced growth rate of cells in biofilms, compared to planktonic cells, increases resistance to antimicrobials that target growth-specific factors. Other mechanisms include the inactivation or degradation of antimicrobials and efflux pumps that remove antimicrobials from the cells [27]. Despite these challenges, ceragenins have been shown to permeate the biofilm extracellular matrix, due to their relatively small size, and eradicate biofilms at relatively low concentrations. This activity is likely

due to the mechanism of action of ceragenins, which is not dependent on the metabolic state of their targets [28].

To quantify the impact of ceragenins on biofilm formation by multidrug-resistant isolates, an XTT assay was performed. The XTT assay measures metabolic activity of cells in the biofilm following a change in color. Corresponding biofilm growth reduction for each strain was calculated compared to a negative control that was not treated with any drugs. As shown in Figure 3, all representative ceragenins demonstrated strong antibiofilm activity against both Gram-positive and Gram-negative multidrug-resistant isolates and caused a substantial reduction of growth. Treatment of *Moraxella* spp., *K. pneumoniae* and *L. pneumophila* decreased biofilm mass by more than 96% compared to the negative control. A previous study showed that in a comparison of CSA-13 with ciproflaxicin, CSA-13 was shown to have greater activity against established biofilms formed by methicillin-resistant *S. aureus* [29].

**Figure 3.** Reduction of established biofilms of ten isolates after 48 h incubation with CSA-131 or/and CSA-44 (100 μg/mL). Using the 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide (XTT) colorimetric based assay, metabolic activity of ceragenin-treated biofilms was measured and the percent of growth reduction was calculated in comparison to an untreated biofilm (control).
