## 2 Results
### Antibiotic film Assays
#### 2.5.1 Minimum Biofilm Inhibitory Concentration (MBIC)  
Since HRZN-15 exhibited strong antibacterial activity and inhibited biofilm formation in the screening assay, it was selected for minimum biofilm inhibitory concentration (MBIC) experiment. _A. buumannii_ strain AB5075 was tested for biofilm formation but did not produce sufficient biofilm mass for the MBIC assay [32]. Instead, _A. buumannii_ BAA-1800 was used in these MBIC assays as it formed more robust biofilms. MBIC measures the lowest concentration of a compound that inhibits bacterial biofilm formation. HRZN-15 was able to inhibit biofilm of _A. buumannii_ BAA-1800 at a slightly higher concentration (8 g/mL, Table 4) than MIC, which is 4 \(\mathrm{\SIUnitSymbolMicro g/mL}\) (Figure S6A), (MBIC\({}_{50}\)\(\sim\)4 \(\mathrm{\SIUnitSymbolMicro g/mL}\)) suggesting that the mechanism is likely bacteria-killing rather than directly inhibiting extracellular polymeric substance (EPS) production. Additional results are presented in Figure S7. LL-37 inhibited _A. buumannii_ BAA-1800 biofilm production at 64 \(\mathrm{\SIUnitSymbolMicro g/mL}\) (Figure S6B) (MBIC\({}_{50}\)\(\sim\)32 \(\mathrm{\SIUnitSymbolMicro g/mL}\)),  
Figure 1: Time-kill kinetics of HRZN-15 and polymysxin B. Inoculum of 5 \(\times\) 10\({}^{5}\) CFU/mL of (**A**,**C**) AB5075 and (**B**,**D**) BAA-1710 were challenged with (**A**,**B**) HRZN-15 at MIC = 8 and 4 \(\mathrm{\SIUnitSymbolMicro g/mL}\) and (**C**,**D**) polymysxin B at MIC = 0.5 \(\mathrm{\SIUnitSymbolMicro g/mL}\) to determine their killing activity over 10, 30, 60, and 180 min. “MIC x” indicates the multiple of MIC used. Bars that are not visible indicate 0 CFU/mL.  
even though it did not inhibit bacterial growth at this concentration, supporting a more direct inhibition of biofilm formation (Figure S7B). This effect has been previously shown for LL-37 against other gram-negative bacteria, like _Pseudomonas aeruginosa_[18; 67]. Peptide IDR-1018 showed a dose-dependent biofilm inhibition of _A. baumannii_ BAA-1800 (Figure S7C) (MBIC\({}_{50}\)\(\sim\)32 \(\mathrm{\SIUnitSymbolMicro g}\)/mL) that was also separate from bacterial growth inhibition (Figure S7C), illustrating that IDR-1018 is able to directly modulate biofilm production separately from its antibacterial activity, likely via its regulation of ppGpp production, as previously reported [66]. Our results for IDR 1018 (Table 4) differ somewhat from [PERSON], 2014 [66], who determined IDR-1018 MBIC\({}_{50}\) = 2 \(\mathrm{\SIUnitSymbolMicro g}\)/mL, MBIC\({}_{100}\) = 10 \(\mathrm{\SIUnitSymbolMicro g}\)/mL, and MIC = 128 \(\mathrm{\SIUnitSymbolMicro g}\)/mL for _A. baumannii_ SENTRY C8 strain, which may reflect strain-dependent differences or broth differences, as they were performed in Basal Medium 2 (BM2), unlike our study in which Tryptic Soy Broth was used. Polymyxin B demonstrated an MBIC of 1 \(\mathrm{\SIUnitSymbolMicro g}\)/mL (Figure S6D), as previously shown against different strains through the biofilm prevention assay [68]. The antibofilm concentration of HRZN peptides is very similar to the antibacterial concentration, illustrating that this peptide can kill MDR _A. baumannii_, even when it is in a biofilm.  
As a side note, it was observed that biofilm mass increases at low peptide concentrations compared to untreated wells, as seen in Figure S6 The mechanism of this effect is unknown but has been observed for other peptides and other bacteria [15; 18; 22; 23].