*3.4. Confocal Laser Scanning of Biofilms*

To visualize antibiofilm properties of ceragenins, biofilms of *Acetobacter* spp. were treated with a lead ceragenin, CSA-131, and prepared for confocal microscopy. Confocal images are shown in Figure 4. In the images, a lack of biofilm is seen in some areas, which could be due to sample preparation in which slides were rinsed prior to staining to remove loosely adhered and planktonic organisms. Overall, as expected, untreated biofilms showed expected aggregates of live cells (green dye, Figure 4A), while ceragenin-treated biofilms exhibited comparable aggregates of dead cells (red dye, Figure 4B). Lack of biofilm was observed more often in the treated than in the untreated cells, which highlights ceragenins' ability to destabilize established biofilms, facilitating their detachment from slide surfaces. Nagent, et al., [9] conducted a biofilm study using confocal microscopy and their images revealed that ceragenins efficiently penetrated established biofilms and led to cell death without significant alterations to the extracellular matrix. Additionally, a recent study demonstrated prolonged inhibition of biofilm formation on endotracheal tube surfaces when the tubes were coated with a CSA-131-containing hydrogel [30].

**Figure 4.** Confocal laser scanning micrographs (×60 magnification) of stained bacterial biofilms. Green: live cells; red: dead cells. (**A**) Untreated *Acetobacter* spp. (**B**) treated with CSA-131 (100 μg/mL).

#### **4. Conclusions**

Obstacles to the development of novel antimicrobial agents include concerns that generation of resistance to one antimicrobial agent may result in cross-resistance to other antimicrobials. Since higher organisms have co-evolved with bacteria, the mechanisms by which they control bacterial growth may provide guidance for development of antimicrobial agents to which bacteria do not readily generate resistance. AMPs represent one of the key means by which higher organisms control bacterial growth. Ceragenins mimic key AMP structural features, specifically, multiple cationic (positive) charges juxtaposed with hydrophobic structure. The studies presented herein demonstrate that even highly multidrug-resistant environmental isolates largely remain susceptible to ceragenins. Additionally, previous studies showed that CSA-13 toxicity is comparable to LL-37 in tested human keratinocytes and it is not toxic to HatCat cells at bactericidal concentrations [31].

The ceragenins tested in this study gave MICs in the single μg/mL range in spite of the high MICs of commonly used antimicrobials, including the last resort antibiotic colistin. SEM images gave results comparable to earlier studies, demonstrating that ceragenins interact with bacterial membranes. Morphological changes to Gram-negative bacterial membranes are a hallmark of the activity of many AMPs, and we have shown, via transmission electron microscopy (TEM) and atomic force microscopy (AFM), that similar changes occur in bacterial membranes upon treatment with a ceragenin [24,32]. Further characterization of the lead ceragenins, CSA-131 and CSA-44, demonstrated that reduction of growth in a preformed biofilm was also successful; however, the extent of reduction was much less compared to the inhibition activity against planktonic cells. Confocal images verified the antibiofilm activity of ceragenins that occurs through penetration of the compound into the extracellular matrix of the biofilm.

Multidrug resistance in Nigeria is on the rise [33]. The highly resistant nature of the Nigerian environmental isolates analyzed in this study suggests that the careful designing and adoption of a multi-sectoral antimicrobial resistance surveillance plan for research and diagnostic purposes should be implemented. Relevant ministries and governmental agencies should consider the following: registration and observation of production premises, particularly where food-producing animals are concerned; improved biosecurity compliance in food-animal environments; banning antibiotic use for animal growth promotion or prophylactic treatment in animal husbandry; implementation of a drug withdrawal period for food animals.

**Author Contributions:** M.M.H. and A.O.M. designed the experiments and wrote the manuscript. B.S.H., J.C., J.W., M.F.T., J.R., D.B., T.S., and S.D. performed the experiments and data analysis. P.B.S. supervised and edited the manuscript. P.B.S. is a paid consultant for N8 Medical and CSA Biotech.

**Acknowledgments:** Generous funding is acknowledged from N8 Medical, Inc., CSA Biotech and Brigham Young University.

**Funding:** This research received no external funding.

**Conflicts of Interest:** The authors declare no conflict of interest.
