Escherichia coli ATCC 25922, P. aeruginosa ATCC 27853, Serratia marcescens ATCC 8100, Salmonella typhimurium ATCC 14028, P. multocida P-1581, Bacillus subtilis ATCC 6633, and S. aureus strains ATCC 25923, ATCC 29213, and T-5706 are maintained as stock cultures in this laboratory. Escherichia coli K12 413 was provided by Dr. Gerwald Köhler (Oklahoma State University Center for Health Sciences, Tulsa, OK). All other Serratia species as well as Bacillus cereus, Staphylococcus epidermidis SK01, Enterococcus faecalis, Lactococcus lactis, and Micrococcus luteus were obtained from Dr. Sue Katz Amburn (Rogers State University, Claremore, OK). Methicillin-resistant Staphylococcus aureus strains SFL8 and SFL64 were obtained from Dr. Sallie Ruskoski (Northeastern State University, Broken Arrow, OK), while Clostridioides difficile strains R20291 and 630 were provided by Dr. George (I-Hsiu) Huang (Oklahoma State University Center for Health Sciences, Tulsa, OK).

Stock cultures were maintained at -80°C in Brain Heart Infusion (BHI; Becton, Dickinson and Co., Sparks, MD) and glycerol (15%) (Darnell et al., 1987) or at 4°C on Mueller Hinton Agar (MHA; Becton, Dickinson and Co.) plates. MHA working cultures were produced by inoculating MHA plates with stock culture cells, incubating at 37°C for about 18 h, and storing at 4°C until needed to inoculate starter cultures consisting of 20 mL volumes of Mueller Hinton Broth (MHB; Becton, Dickinson and Co.) contained in 125 mL screw-capped culture flasks or 5.0-mL volumes contained in Morton closure-capped culture tubes (18 x 150 mm). Stationary-phase inocula for test cultures were obtained by incubating starter cultures at 37°C with rotary aeration at 180 rpm for 12-15 h in an Excella E24 Incubator Shaker (New Brunswick Scientific Co., Edison, NJ). Working and starter cultures of C. difficile strains R20291 and 630 were prepared similarly using Brain Heart Infusion Agar (Becton, Dickinson and Co.) supplemented with 0.5% Difco Yeast Extract (BHIA-Yext; Becton, Dickinson and Co.) and BHI plus yeast extract (0.5%; BHI-Yext). Cultures were incubated at 37°C for 24 h in a GasPak anaerobic jar (Becton, Dickinson and Co.) or a Type A Vinyl Anaerobic Chamber (Model no. 7150000; Coy Laboratory Products, Grass Lake, MI). Oxygen was removed from all media by storing under anaerobic conditions for at least 4 h prior to use.

Melanin-inspired compounds designated EIPE-HCl (hydrophilic) and EIPE-1 (hydrophobic) ( Figure 1 ) were synthesized in the laboratory of Dr. Toby L. Nelson as described elsewhere (Selvaraju et al., 2015; Selvaraju et al., 2016; Adhikari et al., 2022). Stock solutions were prepared at desired concentrations in dimethyl sulfoxide (DMSO; Fisher Scientific, Fair Lawn, NJ) and stored in microcentrifuge tubes at 4°C until needed.

Disparate bacterial pathogens were screened for susceptibility to EIPE-HCl and EIPE-1 using a standardized disk agar diffusion bioassay as described by Clayborn et al. (2011). EIPE-HCl and EIPE-1 stock solutions (2.5 µg/µL each) were prepared in DMSO using vortex agitation. Sterile blank filter disks (6.0-mm diam; Becton Dickinson and Co.) were infused with DMSO (10 µL; control), EIPE-HCl (25 µg in 10 µL DMSO), or EIPE-1 (25 µg in 10 µL DMSO) to yield compound potencies of 25 µg per disk. Disks were dried under flowing sterile air in a biosafety cabinet before storing at 4°C until needed. Overnight starter cultures were diluted into fresh MHB to prepare test culture inocula at an OD₆₂₀ of 0.025 using a Spectronic 20D optical spectrophotometer (Thermo Electron Corp., Madison, WI). Loaded disks were applied to MHA plates uniformly seeded with standardized test culture inocula and allowed to sit at 4°C for 1.0 h to allow compound diffusion in the absence of bacterial growth prior to aerobic incubation for 18 h at 37°C. C. difficile strains were uniformly spread onto BHIA-Yext plates and incubated anaerobically for 24 h at 37°C. Electronic calipers (Fisher Scientific, Waltham, MA) were employed to measure growth inhibition zone diameters from which disk diameters were subtracted.

Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) values were determined for EIPE-1 against disparate bacterial pathogens using our modification of a conventional microbroth dilution bioassay (Adhikari et al., 2022). EIPE-1 was dissolved in DMSO to a concentration of 2,560 µg/mL before diluting into sterile MHB to yield a final concentration of 128 µg/mL. Starter culture cells were used to inoculate MHB test cultures to an OD₆₂₀ of 0.01 with the aid of a Spectronic 20D optical spectrophotometer. MICs were determined in microtiter plates (Fisherbrand 96 DeepWell Polypropylene Microplates; ThermoFisher Scientific, Waltham, MA) using a conventional two-fold dilution scheme beginning with an EIPE-1 stock concentration of 128 µg/mL. Assay microtiter plates were covered with an aluminum seal (Nunc Aluminum Seal; ThermoFisher Scientific, Waltham, MA) and incubated for 24 h at 37°C and 180 rpm in an Excella E24 Incubator Shaker. BHI-Yext was employed in place of MHB for C. difficile strains which were incubated anaerobically for 24 h at 37°C. MIC values were determined based on the lowest EIPE-1 concentration that completely inhibited the initiation of growth as judged by visualizing the absence of turbidity in each dilution series. MBCs were performed by transferring a small volume from each well onto a MHA plate using a 48-prong plate stamper and incubating at 37°C for 18 h. BHIA-Yext plates were employed for C. difficile strains which were incubated anaerobically at 37°C for 24 h. MBC values were determined on the basis of the lowest EIPE-1 concentration that completely inhibited macroscopic colony formation.

The effect of EIPE-1 on the batch cultural growth kinetics of selected gram-negative and gram-positive bacterial pathogens was determined using a method we have previously described (Champlin et al., 2005; Ellison and Champlin, 2007; Ellison et al., 2007) with the following modifications. EIPE-1 stock solutions were prepared in DMSO at concentrations of 100 µg/mL and 1000 µg/mL. Sterile MHB was inoculated with starter culture cells to an OD₆₂₀ of 0.05 to provide test cultures. Aliquots of 5.0 mL each were transferred to culture tubes (18 x 150-mm diameter; Kimax with Morton closures). Each culture was treated with 1.0 µL of the appropriate stock solutions to reach final EIPE-1 concentrations of 0.2 µg/mL and 0.02 µg/mL, respectively. A solvent control was prepared by treating one test culture with 1.0 µL of DMSO to yield a final concentration of 0.02% (v/v), while another was left untreated to serve as a positive growth control. Excella E24 Incubator Shaker incubation was carried out at 37°C and 180 rpm while batch cultural growth was monitored turbidimetrically (OD₆₂₀) at 30-min intervals using a Spectronic 20D optical spectrophotometer.

Efforts to sensitize selected gram-negative pathogens to EIPE-1 by chemically permeabilizing their outer membranes was attempted using the outer membrane permeabilizer compound 48/80 (Katsu et al., 1985) as described by Champlin et al. (2005). Batch test cultures were prepared and cultivated as described above after treatment with outer membrane permeabilizer compound 48/80 (5.0 µg/mL; Sigma-Aldridge Chemical Co., St. Louis, MO) and EIPE-1 (0.2 µg/mL) both alone and in combination to detect synergistic inhibition of batch cultural growth. The known susceptivity of P. aeruginosa to compound 48/80 sensitization to triclosan (Champlin et al., 2005; Ellison et al., 2007) was employed as a positive control. A triclosan (Irgasan DP 300; Ciba Specialty Chemical Corp., High Point, NC) stock solution was prepared in ethanol (95%) at a concentration of 2.0 µg/mL.

The screening of 14 strains of five gram-negative and 13 strains of eight gram-positive pathogenic bacteria for susceptibility to EIPE-HCl and EIPE-1 was accomplished using a standardized disk agar diffusion bioassay (Clayborn et al., 2011). Potential intrageneric disparity was examined by including multiple species of the gram-negative genus Serratia and gram-positive genus Staphylococcus. As can be seen in Table S1 , all gram-negative organisms were observed to be resistant to both EPIE-HCl and EIPE-1 at a potency of 25 µg/disk. Despite also being resistant to EIPE-HCl, each of the gram-positive organisms was susceptible to EIPE-1 at the potency tested except for B. cereus, E. faecalis, and C. difficile R20291. The magnitude of values obtained with disk potencies of 25 µg was small, yet reproducible. Only slightly larger zones of inhibition were observed when selected organisms were tested with disk potencies as high as 250 µg/disk ( Figure 2 ).

All gram-negative and gram-positive organisms were resistant to the hydrophilic EIPE-HCl derivative at the screening potency of 25 µg/disk. These data suggest the absence of any antibacterial properties rather than an underlying role for cell envelope differences in ultrastructure between gram-negative and gram-positive bacteria with regard to intrinsic resistance. In contrast, most gram-positive bacteria were susceptible to the hydrophobic EIPE-1 while all gram-negative bacteria were resistant thereby suggesting a gram-positive spectrum of antibacterial properties. This conclusion is consistent with gram-negative bacteria being intrinsically resistant to EIPE-1 due to the impermeability of the intact outer membrane to hydrophobic substances in general (Nikaido, 2003; Champlin et al., 2005). The notion that outer cell envelope ultrastructure underlies gram-negative resistance is supported by the fact that P. multocida P-1581, which possesses an outer membrane that is atypically permeable to hydrophobic molecules (Ellison and Champlin, 2007) was slightly susceptible at a potency of 250 µg/disk (data not shown). The disk agar diffusion bioassay is a qualitative screen and cannot be used to precisely establish the efficacy of EIPE-1. This limitation was compounded by the poor diffusion properties suggested by the small magnitude of inhibition zone diameters and the absence of dose dependency on EIPE-1 disk potency ( Table S1 , Figure 2 ). These data suggested that quantitative MIC and MBC values generated by a serial broth dilution bioassay would be necessary to circumvent the apparent poor diffusibility of EIPE-1 to better establish the disparate susceptibility levels of gram-positive organisms.

The quantitative determination of the EIPE-1 MIC and MBC values for selected gram-negative and gram-positive pathogenic bacteria employed a conventional microbroth dilution bioassay (Adhikari et al., 2022). Data contained in Table 1 reveal EIPE-1 MICs and MBCs of 128 µg/mL or greater for all gram-negative bacteria examined. In contrast, much lower values were obtained for gram-positive organisms with MICs ranging from 2.0 to 0.25 µg/mL with the exception of the two strictly anaerobic C. difficile strains for which the MIC and MBC values were two to six fold higher. This was possibly affected by physiological differences which required that the anaerobic organism be cultured on a richer growth medium under anaerobic conditions. C. difficile R20291 and E. faecalis exhibited much lower MIC and MBC values than gram-negative organisms in contrast to both of these gram-positive organisms exhibiting resistance in the standardized disc agar diffusion bioassay further showcases the limitation of poor diffusibility EIPE-1 possesses in agar media. Interestingly, the MBC for S. aureus SFL 64 was considerably greater than the corresponding MIC which is consistent with the enhanced refractory nature of MRSA strains. The uniformly low MIC values for gram-positive organisms in contrast with the uniformly large MIC values obtained for gram-negative bacteria further support the notion that the intrinsic EIPE-1 resistance seen in the latter group is due at least part to outer membrane exclusivity for hydrophobic solutes (Nikaido, 2003; Champlin et al., 2005). The phylogenetic disparity of the gram-negative organisms supports this conclusion because gram-negative cell envelope ultrastructure is their most notable phenotypic congruity likely to be involved in an antibacterial agent resistance mechanism.

The effect of EIPE-1 on the growth kinetics of susceptible gram-positive pathogens was determined by administering EIPE-1 to batch cultures of selected organisms. This approach allowed for circumvention of the apparent poor diffusibility of EIPE-1 in agar-solidified growth media. Growth curves were constructed to initially assess the cellular and molecular mechanisms by which the compound is able to inhibit gram-positive bacterial growth. EIPE-1 concentrations of both 0.02 and 0.2 µg/mL failed to inhibit the growth of any of the five gram-negative organisms examined ( Figure 3 ) including two cell surface hydrophobicity variants of P. multocida (Darnell et al., 1987). Because the outer membrane of P. multocida is atypically permeable to hydrophobic substances (Ellison and Champlin, 2007), this observation supports the conclusion that intrinsic resistance to EIPE-1 in gram-negative bacteria may involve an ancillary mechanism other than simple outer membrane exclusion of hydrophobic substances. Delcour (2009) posited that intrinsic antimicrobial resistance in gram-negative bacteria can be due to synergy between outer membrane impermeability and the presence of active efflux pumps, a relationship we previously demonstrated in P. aeruginosa (Ellison et al., 2007).

Each of the five gram-positive organisms examined were susceptible to different degrees when treated with EIPE-1 at a concentration of 0.2 µg/mL in batch cultures ( Figure 4 ). B. subtilis ATCC 6633 was the most susceptible and can be seen to undergo bacteriolysis at the time of treatment. EIPE-1 was also bacteriolytic at this concentration for S. epidermidis SK01 although at a considerably slower rate. The growth rates of S. aureus ATCC 25923 and SFL 64 were markedly decreased while E. faecalis and S. lactis underwent only growth rate depressions in the presence of EIPE-1 at this concentration. Greater concentrations were not examined in this experiment for the purpose of minimizing potential secondary effects.

The intrinsic resistance of all the gram-negative organisms to an EPIE-1 concentration that was either growth rate inhibitory or bacteriolytic for all of the gram-positive organisms confirms its gram-positive antibacterial specificity. Moreover, the rapid bacteriolysis of B. subtilis ATCC 6633 suggests that EIPE-1 acts to inhibit the growth of susceptible bacteria by means of a cytoplasmic membrane-directed mechanism of action. The slower lysis rate observed in S. epidermidis SK01 would need to be studied further to validate that EIPE-1 is targeting its cytoplasmic membrane and not an anabolic cell process.

Both B. subtilis ATCC 6633 and S. epidermidis SK01 were extremely susceptible to EIPE-1 with MICs in the 0.25 to 1.0 µg/mL range ( Table 1 ). In order to further investigate the underlying mechanism by which EIPE-1 is able to exert its antibacterial properties on gram-positive organisms, batch cultures were titrated with a serial two-fold dilution ( Figure 5 ). B. subtilis ATCC 6633 underwent rapid bacteriolysis with 0.1 and 0.2 µg/mL concentrations, while only slight growth rate inhibition was seen at 0.05 µg/mL suggesting that a distinct concentration threshold must be achieved at which bacteriolysis is affected. In contrast, S. epidermidis SK01 was susceptible to all EIPE-1 concentrations in a dose-dependent manner with bacteriolysis only occurring with the highest concentration of 0.2 µg/mL.

In view of the hydrophobic nature of EIPE-1, its gram-positive antibacterial spectrum strongly suggests that the intrinsic resistance seen in disparate gram-negative pathogens is due to exclusion by virtue of the impermeability properties of their outer cell envelopes for nonpolar molecules. To test this possibility more specifically, the outer membrane permeabilizer compound 48/80 was administered to chemically disrupt the cell surfaces of selected gram-negative pathogens (Katsu et al., 1985; Champlin et al., 2005) in an effort to synergistically sensitize the organisms to EIPE-1. Data contained in Figure 6A show only slight to no synergistic sensitization due to treatment with compound 48/80 and EIPE-1 in any of the three gram-negative test bacteria at a permeabilizer concentration that effectively sensitized P. aeruginosa to the hydrophobic biocide triclosan ( Figure 6B ). The absence of synergy between outer membrane permeabilization and EIPE-1 for these three gram-negative bacteria indicates that intrinsic EIPE-1 resistance in gram-negative organisms cannot be explained solely based on outer membrane impermeability to hydrophobic molecules and suggests the presence of an ancillary intrinsic resistance mechanism.