*2.1. Isolation and Maintenance of Bacterial Isolates*

Bacteriological analyses of water samples, including heterotrophic plate count (HPC), fecal coliform (FC) and total coliform count (TCC), were determined using both the direct pour plate method and membrane filtration techniques. No serial dilution was carried out. Nutrient agar medium was used for heterotrophic bacteria plate counts. MacConkey agar (MCA) was used for total coliform counts, and membrane fecal coliform (MF-C) agar medium was used for fecal coliform counts. The plates were inoculated in triplicate. Inoculations for HPC and TCC were conducted by adding 1 mL of sample to each plate. For membrane filtration, 100 mL of each water sample was filtered through a 0.45 μm membrane filter before aseptic transfer of the membrane onto MF-C agar or MCA for FC and TCC respectively. No dilution was used in either method. MCA was used for isolation of lactose fermenters (coliforms).

For isolates taken from plants, natural rubber latex (1 mL) was added to sterile distilled water (9 mL) and then serially diluted using a sterile micropipette. One gram of deteriorated rubber latex was added to sterile distilled water (100 mL) in a conical flask. The combination was mixed well, and an aliquot from this mixture was serially diluted in water (10−<sup>1</sup> to 10−<sup>10</sup> dilution). Selected dilutions were then used for the inoculation of agar plates. Each sample was plated in triplicate using the pour plate method. Inoculated plates were incubated for 24 h at 35–37 ◦C. Colonies were enumerated using a colony counter for total heterotrophic bacteria and total coliform counts. Discrete colonies were sub-cultured onto fresh nutrient agar plates aseptically to obtain pure cultures of the isolates and were stored in a refrigerator at 4 ◦C for further identification.

#### *2.2. Identification of Isolates*

Bacterial isolates were characterized based on microscopic appearance, colony morphology, gram staining reactions, and appropriate biochemical tests based on Bergey's Manual of Determinative Bacteriology and as described by Cheesbrough [19]. The isolates were identified by comparing their characteristics with those of known taxa, as described by Cruickshank et al. [20] and Holt [21]. Table 1 shows the source of ten isolates used in this study.


**Table 1.** Isolation source of bacteria used in this study.

#### *2.3. Susceptibility Testing*

Minimum inhibitory concentrations (MICs) were determined using a broth microdilution method in a 96-well microdilution plate according to the Clinical Laboratory Standards Institute protocol [22]. Briefly, 96-well plates were prepared with individual wells containing doubling concentrations of selected ceragenins, including CSA-13, CSA-44, CSA-131, and CSA-144 in the appropriate culture medium for a total volume of 100 μL. A selection of commercial antimicrobials including chlorhexidine, kanamaycin, colistin, polymyxin B, erythromycin, tetracycline, vancomycin and ampicillin was also used for comparison. An inoculation of 100 μL at 10<sup>6</sup> CFU/mL was added to each well. Each of the 10 isolates was tested in duplicate and each plate contained positive and negative controls. Plates were incubated for 24 h at 37 ◦C. After incubation, results were obtained by examining wells for turbidity. Minimum bactericidal concentrations (MBCs) were determined by taking 10 μL from each well and plating on agar media. The MBC was defined as the lowest concentration of an antibacterial agent giving no visible colonies after 24 h incubation at 37 ◦C [10].
