Evaluation of a 2-aminoimidazole variant as adjuvant treatment for dermal bacterial infections

Figures & data

Figure 1 Biofilm inhibition assay for H10 against S. aureus and P. aeruginosa.

Notes: Inhibition assay performed to determine the working concentration for H10 in S. aureus and P. aeruginosa. The data are given as the mean ± SD. (Inset) Chemical structure of 2-amino-N-undecyl-H-imidazole-5-butanamide (H10).
Abbreviations: SD, standard deviation; S. aureus, Staphylococcus aureus; P. aeruginosa, Pseudomonas aeruginosa.

Figure 2 Swine skin dosing and histology schedule.

Notes: Doses (represented by ℞) of H10 (5 mM) were administered to all test sites at 0 hour (day 0). After 24 hours, another dose was administered to all sites. On day 2, another dose was given to all sites except “48 hours”, which was imaged, biopsied, closed, and sealed (represented by ☤). On day 3, a final dose was administered for “168 hours”, while “72 hours” was imaged, biopsied, closed, and sealed. Finally, after 7 days, the final “168 hours” site was examined.

Figure 3 Growth curves for S. aureus and P. aeruginosa in the presence of H10.

Notes: Representative growth curves for S. aureus (light gray lines) and P. aeruginosa (dark gray lines) in the presence of H10 at a concentration greater than the IC₅₀. Data are presented as the mean ± SD of each group. The boxes and circles represent the control values and treatment with H10 at the indicated concentration, respectively.
Abbreviations: IC₅₀, half maximal inhibitory concentration; SA, Staphylococcus aureus; PA, Pseudomonas aeruginosa; SD, standard deviation.

Figure 4 Preformed biofilms are dispersed in the presence of H10.

Notes: Viable cell counts of S. aureus and P. aeruginosa 3-day biofilms following treatment with H10 (24 hours), showing dispersion of a preformed biofilm. The data are given as the mean ± SD, n=3. The asterisk (*) indicates values with significant difference from the other group within the species (P≤0.020).
Abbreviations: CFU, colony-forming unit; SA, Staphylococcus aureus; PA, Pseudomonas aeruginosa; SD, standard deviation.

Figure 5 Synergism of H10 with antibiotic cocktail in S. aureus.

Notes: When S. aureus is grown in a combination of H10 and a P/S cocktail, the antibiotic-resistant S. aureus 29213 strain becomes resensitized to antibiotic treatment. The data are given as the mean ± SD, n=3. Statistically significant differences between columns (Tukey’s HSD intervals at P<0.05) are marked with different letters (a–c).
Abbreviations: CFU, colony-forming unit; P/S, penicillin/streptomycin; S. aureus, Staphylococcus aureus; SD, standard deviation; HSD, honest significant difference.

Figure 6 hFF viability assay in BCM.

Notes: In hFF cells, BCM from S. aureus grown in the presence of H10 (BCM + H10) does not exhibit the deleterious effects of S. aureus BCM. The data are given as the mean ± SD, n=3. The asterisk (*) indicates statistically significant difference from both the control and BCM + H10 (P≤0.002).
Abbreviations: BCM, biofilm-conditioned medium; hFF, human foreskin fibroblast; S. aureus, Staphylococcus aureus.

Figure 7 Percent scratch area closed for the in vitro scratch assay.

Notes: Results are shown for two experimental groups, where hFFs were exposed to either antibiotic-resistant Staphylococcus aureus BCM or BCM from these cells grown in the presence of H10 (BCM + H10). The results for one control group are also shown. The data are given as the mean ± SD, n=4. The asterisk (*) indicates values with significant difference from the other groups at the same time point (P≤0.001).
Abbreviations: hFFs, human foreskin fibroblasts; BCM, biofilm-conditioned medium; SD, standard deviation.

Table 1 Histopathological evaluation of H10 epidermal/dermal toxicity

Condition	Endpoint	Replicate	Epidermal histopathology						Dermal histopathology
			Inflammation		Hydropic		Necrosis		Inflammation		Edema		Hemorrhage
			E	L	E	L	E	L	E	L	E	L	E	L
25% P407 control		1	0	0	0	0	0	0	1	1	0	0	0	0
		2	0	0	0	0	0	0	0	0	0	0	0	0
20% SDS in EtOH		1	0	0	0	0	0	0	2	2	0	0	0	0
		2	0	0	0	0	0	0	1	1	0	0	0	0
5% DMSO in 25% P407		1	0	0	0	0	0	0	0	1	0	0	0	0
		2	0	0	0	0	0	0	0	0	0	0	0	0
5 mM H10 in 25% P407 48 hours		1	0	0	0	0	0	0	0	1	0	0	0	0
		2	0	0	0	0	0	0	0	0	0	0	0	0
5 mM H10 in 25% P407 72 hours		1	0	0	0	0	0	0	0	0–1	0	0	0	0
		2	0	0	0	0	0	0	0	1	0	0	0	0
5 mM H10 in 25% P407 168 hours		1	0	0	0	0	0	0	0	0	0	0	0	0
		2	0	0	0	0	0	0	0	0–1	0	0	0	0

Notes: Effects of acute dermal exposure to H10 in a swine model. Histopathology slides prepared from punch biopsies were scored for the presence of inflammatory cells on a Draize scale of 0–4, where 0= absent, 1= minimal, 2= mild, 3= moderate, and 4= marked. Duplicate doses were split across two animals, and the image included for each condition represents the photograph taken at the end of treatment, prior to biopsy.

Abbreviations: E, eosinophils; L, lymphocytes; P407, poloxamer 407; SDS, sodium dodecyl sulfate; EtOH, ethanol; DMSO, dimethyl sulfoxide.