A showed the antibacterial behavior of the novel AMPs against S. aureus
at five different time intervals. All of the tested AMPs had good and stable antibacterial effects against S. aureus
. During the 24 h of incubation, (3) had a relatively higher efficacy, as it could reach more than 99.99% inhibition from the beginning of the test. Furthermore, (2) and (1) showed high efficacies during the test period, with more stable antibacterial effects. Lastly, (4) showed a relatively lower effect compared to (3), (1), and (2). As described in Figure 3
B, (2) showed the maximum antibacterial effect against P. aeruginosa
PA14 at double the MIC value. Additionally, its antibacterial activity was unwavering over the whole period of incubation, as it affects more than 99.99% of the bacteria from the beginning of the incubation. In terms of efficiency, (1) come next to (2), showing a remarkable antibacterial effect, as it affected more than 97% of the bacteria from the start of the reaction. In comparison with (1) and (2), (4) and (3) exhibited slightly lower efficacies, as they started by inhibiting more than 95% and 92% of the bacterial growth, respectively, while their full effect was only exerted after 24 h of incubation. As illustrated in Figure 3
C, (2) and (1) had the most active and stable antibacterial pattern. They could affect more than 97% of the bacteria at 0 h of incubation. Furthermore, (2) was more effective against E. coli
than (1) and other peptides. However, (3) and (4) showed the maximum antibacterial effect at 24 h of incubation, and the bacteria showed little resistance against them. As shown in Figure 3
D, (2) was the most potent AMP against B. subtilis
, with a stable antibacterial effect over the examination time, except after 4 h. However, after 8 h, the peptide could overcome the bacterial resistance, reaching more than 99.99% inhibition of the bacterial growth. Additionally, (1) was the second peptide in efficacy, as it could overcome the bacterial resistance after 4 h of incubation. However, the bacteria could resist the antibacterial effect of (4) and (3) from 4 h until 18 h. After that, they could inhibit more than 95% and 99.99% of the bacteria, respectively.
The results described in Figure 4
show the inhibition of the bacterial growth in the tested samples compared with the controls before incubation. We can see that (2) showed relatively high activity (except for S. aureus
), as it could inhibit the growth of P. aeruginosa
and B. subtilis
completely, while the %growth of the other bacteria was about 3%. According to the activity, (1) came in second place, except for against S. aureus
, for which it showed the same efficacy as (2).In addition, (3) came in third place according to its activity against all bacteria except for S. aureus
; it was the most active peptide, as its associated bacterial growth was about 2%. While (4) showed a good antibacterial activity, it was relatively lower than the other peptides except for P. aeruginosa
; it was more active than (3).
The antibacterial property of the cyclic peptide (2) fares better compared with (1). This higher activity depends on the more rigid structure and the higher hydrophobicity of the cyclic conformation, allowing (2) to penetrate the bacterial cell more effectively than the linear peptide, causing a more inhibitory effect on the bacterial growth. Replacing the tyrosine residue with a histidine slightly improved the overall antibacterial effect of (3) and (4) over that of the (1) and (2) peptides. We suggest that the increase in the antibacterial effect is due to the positively-charged histidine residue located in the center of the two phenylalanine residues, which creates a relatively stronger attacking nucleus. This nucleus has more penetrating ability into the bacterial cell membrane than that of the tyrosine nucleus. Besides this, the secondary structure created by (3) can penetrate the bacterial cell membranes more rapidly than its cyclic form.
The cell-penetrating abilities of (3) and (4) were slightly augmented by the electrostatic interaction of the positively-charged histidine residue with the negatively-charged bacterial membranes. We posited that this interaction occurred during the penetration of the hydrophobic sidechain groups surrounding the histidine moiety. These double interaction forces increase the penetration rate of the peptide, causing the higher inhibition of bacterial cell growth. In addition, the Phe-His-Phe nucleus showed a better antibacterial effect in the linear form than that of the cyclic peptide, except in the case of B. subtilis bacterial strain. We assume that this nucleus penetrates the bacterial cell membrane more efficiently when it is connected to a free linear structure rather than the cyclic rigid form (i.e., the secondary structure of the linear form was more active and flexible than that of the cyclic form). B. subtilis is one of the spore-forming bacterial strains in which the rigid cyclic structure showed more penetrating ability into these persister cells than the linear form. The time-inhibition assays suggested a concentration-dependent, rather than time-dependent, inhibition of the bacteria by the tested peptides.