*3.1. Susceptibility of Isolated Bacteria*

In an initial set of experiments, the MICs of several common antimicrobials against ten bacterial isolates were determined (Table 2). Gram-positive bacteria, including *Actinomycins* species, were relatively susceptible to chlorhexidine, vancomycin and ampicillin; however, all tested Gram-negative isolates showed very high MICs with selected antimicrobials, including chlorhexidine, kanamycin, colistin, polymyxin B, erythromycin and tetracyclin. For example, the MIC of *Enterococcus* species with chlorhexidine, kanamycin, colistin, polymyxin B, erythromycin and tetracyclin was 64, 100, >100, 100, 8 and 32 μg/mL, respectively. Of particular note is the MIC of colistin, which was more than 100 μg/mL with most of isolates tested. Colistin is generally considered the therapeutic of last resort for multidrug-resistant Gram-negative bacterial infections, so the presence of highly colistin-resistant isolates in this study is alarming.


**Table 2.** Minimum inhibitory concentrations (MICs) (μg/mL) of ten isolates with common antibiotics.

Chl: chlorhexidine; Kan: kanamycin; Col: colistin; Pol B: polymyxin B; Ery: erythromycin; Tet: tetracycline; Van: vancomycin; Amp: ampicillin. nm: not measured.

To determine the activity of ceragenins against multidrug-resistant isolates, the MICs of selected ceragenins CSA-13, CSA-44, CSA-131 and CSA-144 were measured. These results are shown in Table 3. Ceragenins retained activity against all multidrug-resistant strains and showed low MICs compared to the commonly used antimicrobials. The MIC of CSA-13 and CSA-131 of 1–2 μg/mL with all of the highly multidrug-resistant isolates was of particular note. This result is consistent with our previous studies showing that ceragenins are highly active against methicillin-resistant *Staphylococcus aureus* [9] and colistin-resistant *Klebsiella pneumoniae* [10].

**Table 3.** Comparison of the MIC (minimum bactericidal concentrations (MBC)) (μg/mL) of ten isolates to selected ceragenins.


To confirm that the activity of ceragenins is bactericidal, MBCs of the same ceragenins were measured with the multidrug-resistant isolates. All tested ceragenins were found to be bactericidal at a range of 1–100 μg/mL, exhibiting bactericidal activity against strains such as *Pseudomonas* and *Actinomyces* spp. at the same concentrations as the corresponding MICs, suggesting that antibacterial activity of ceragenins are likely bactericidal rather than bacteriostatic.
