*3.4. Antimicrobial Activity*

The two peptide conjugates (Table 1) were intended to mimic and/or enhance the activity of the parent IDR1018 while improving biocompatibility and physicochemical properties. Despite an enhancement or retention of immunomodulatory activities, there was a 2 to 8-fold reduction in antimicrobial activity (i.e., higher MIC values) when compared to the parent IDR1018 (Table 2).


**Table 2.** Activities of the peptide conjugates vs. planktonic bacteria.

The MICs of the PEG6-IDR1018 ranged between 32–64 μg mL<sup>−</sup>1, while the MICs of the Glc-IDR1018 ranged between 64–128 μg mL−<sup>1</sup> based on the strains tested. According to the literature, [26,29,43,49,50], such decreases in antimicrobial activity are not uncommon for PEGylated and glycosylated peptides. The activity of the antimicrobial peptide Nisin, for instance, was reduced by conjugation with polyethylene glycol [50]. Likewise, the activity of other antimicrobial HDPs, such as α defensine 1 [51], tachyplestin I [52], magainin 2 [49], aurein 2.2 [53] and others [26], all exhibited reduced antimicrobial activity when pegylated. It is worth noting that previous results for pegylation and glycosylation appear to depend on the type of ligation, the PEG or glycan composition and structure, the nature of the PEG or the sugar-peptide bond and their lengths, and the overall structural conformation of the generated molecules [54,55]. In a study by Falciani C et al., for instance, pegylation at the C-terminus of the antimicrobial peptide M33 was shown to improve the stability of the peptide against *Pseudomonas aeruginosa* elastase [56]. While on the contrary, conjugation at the N-terminus of HDPs, LL-37 and cecropin A, derivatives (PEG-CaLL), showed to improve the antimicrobial activity against various bacterial strains, including *B. anthracis* (including vegetative and spore forms), *Escherichia coli* and *Staphylococcus aureus* when compared to the LL-37 peptide [57]. By analogy, in a study by Talat et al., the activity of the glycosylated peptides was shown to be impacted by the stereochemistry of attached sugars [58]. The study also showed that β-linked sugars induce more flexible and conformationally unstrained conjugates in contrast to the α-linked counterparts which cause more rigid and highly stable conjugates [58]. Although the N and C termini of peptides tend to show conformational flexibility, further studies would be required to investigate if these conjugations impacted the overall HDP IDR1018 chemical structure including its secondary structure, in order to understand the potential reasons behind the reduced activity. It would also be worthwhile investigating the impact of these conjugations on other antimicrobial HDPs, including IDR1018 analogs.

### *3.5. Antibiofilm Activity*

Effects on bacterial biomass were first tested using a crystal violet staining assay. While none of the peptides exhibited complete eradication of the biomass staining in the wells at the MIC values, the results showed a reduced ability of the designated conjugates to reduce the adhered biofilm mass by >90%, at much higher concentrations compared to the original IDR1018 (Figure 4). The best antibiofilm activity of the conjugates was observed against *S. aureus* MRSA0017, where ≥90% of the biomass reduction was observed at <sup>64</sup> <sup>μ</sup>g mL−<sup>1</sup> for both PEG6-IDR1018 and Glc-IDR1018 peptides. The results were supported by determining the viable biofilm cell counts (CFU) following biofilm treatments in the biofilm eradication assay (where biofilms were pre-formed), indicating comparable outcomes to the biomass dispersal results (Table 3). The peptide conjugates exhibited a 3 to 16-fold reduction in

the antibiofilm activity (i.e., higher minimal biofilm eradication concentration values or MBEC = 64–256 μg mL<sup>−</sup>1) compared to the unmodified IDR1018 (MBEC = 16 μg mL<sup>−</sup>1) for all tested strains. Thus, in agreement with the MIC results, the pegylation and glycosylation led to maintenance but a weakening of the antibiofilm properties of the IDR1018. It is clear from these results that pegylation and glycosylation modifications did not generate more efficacious antimicrobial or antibiofilm peptides and that their influences on the HDPs' antimicrobial and antibiofilm activity seemed peptide-specific.

**Figure 4.** Effect of the peptide conjugates in reducing bacterial biofilm mass as assessed by the crystal violet staining assay. (**a**–**d**) show the antibiofilm activity against *S. aureus* MRSA0017, *P. aeruginosa* PA14, *E. coli* ATTC489, and *A. baumannii* 5015, respectively. The % of biofilm mass reduction was determined relative to untreated samples. The data represents the mean values ± standard deviation. All experiments were conducted three times. The green and grey shades indicate relative MBECs.


**Table 3.** Activities of the peptide conjugates vs. preformed bacterial biofilms (minimal biofilm eradication concentrations, MBECs).
