Mh broth

Cultures of a media-adapted wild-type E. coli MG1655 were grown from independent single colonies overnight at 37 °C in Mueller–Hinton (MH) broth (Becton-Dickson). From each culture, 100-µL aliquots were spread onto five MH agar plates containing different ciprofloxacin concentrations, 0.032, 0.064, 0.096, 0.128, and 0.16 µg/mL, corresponding to 2, 4, 6, 8, and 10 times the MIC of the parental strain. Plates were incubated for up to 48 h and examined for the growth of colonies. From each culture a single colony was picked from the highest drug concentration where growth occurred (defined as colony diameter ≥1 mm within 48 h). Each colony was purified by streaking on MH agar plates with the drug concentration at which it was isolated. Second-step mutants were selected by resuspending a colony of each of the purified first-step mutants in 0.9% NaCl and plating ∼10⁸ colony-forming units onto MH agar plates with ciprofloxacin at 2, 4, 6, 8, and 10 times the concentration at which the mutant was originally selected and purified. Selection was continued through 3–8 successive cycles until the concentration at which mutants were being selected reached or exceeded 1 mg/L.

The antimicrobial activity of the bio-produced pexiganan peptide was determined as compared to that of the controls (water, DAMP4 protein, DAMP4_var-pexiganan protein, and synthetic pexiganan peptide) by using the minimum bactericidal concentration (MBC) method (Hu et al. 2010 (link)). Briefly, a single colony of E. coli ATCC^® 25922™ (Manassas, VA) selected from a freshly streaked plate was inoculated into 5 mL Mueller-Hinton (MH) Broth (Becton–Dickinson, Sparks, MD) at 37 °C, 180 rpm, and then harvested at the exponential growth phase (OD₆₀₀ ~ 0.5). After rinsing the cells twice by centrifugation (4000×g, 4 °C, 20 min), a standard cell suspension was prepared by resuspending the cell pellet in 0.9% NaCl solution to a final concentration of 10⁷ colony-forming units (CFU) per mL (OD₆₀₀ ~ 0.08). Protein/peptide samples (at final concentrations ranging from 1 to 32 µg/mL) as well as water were added into the standard cell suspensions to a final volume of 2 mL. Following incubation at 37 °C, 180 rpm for 2 h, the mixtures (diluted 10,000× in sterilized water) were each spread onto MH agar plates (MH Broth, 1.5% agar) and then incubated at 37 °C for overnight. The percentages of viable cells grown on the agar plates containing the protein/peptide samples were determined by counting the number of the colonies in comparison with the controls.

The reference strain C. jejuni ATCC 33560 and a field strain of C. coli isolated from chicken meat were used to assess the growth kinetics. One ESBL-producing E. coli strain carrying the CTX-M-15 gene (Accession No. MH756636) was isolated from chicken meat previously in our lab and the gene was confirmed via sequencing. The Campylobacter strains were maintained at −70°C in Bolton broth (Oxoid Ltd., Basingstoke, England) with 10% horse blood (Oxoid) and 20% glycerol, and the stock was plated on 5% sheep blood agar plates (Komed, Seongnam, South Korea) at 41.5°C for 48 h under microaerophilic condition (10% CO₂, 5% O₂, and 85% N₂). The E. coli stock was maintained in Tryptone Soy broth (Difco, Maryland, USA) and plated on MacConkey agar (Difco, Maryland, USA). Subsequently, single colonies were suspended in MH broth (Becton, Maryland, USA) to obtain stationary phase cultures, and bacterial concentrations of Campylobacter and E. coli were determined via serial dilution and plating on blood agar with 48 h incubation and MacConkey agar with 24 h incubation, respectively.

To determine the minimal inhibitory concentration (MIC) of KYE28, a microtiter broth dilution method was utilized, as previously described in the NCSLA guidelines [32] (link). In brief, overnight cultures of indicated bacteria were suspended and further diluted in Mueller-Hinton (MH) broth (Becton, Dickinson). KYE28 was dissolved in H₂O to concentrations ranging from 320 µM to 2.5 µM by serial dilution. Fifty µL of each concentration was added to the corresponding well of a 96-well microtiter plate (polypropylene, Costar Corp.) together with 50 µL of bacteria (2×10⁵) in MH broth. The plate was incubated at 37 °C for 16–18 h. The MIC was obtained as the lowest concentration where no visual growth of bacteria was detected.

The bacterial isolates used in this study included 12 pairs of DAP^S and DAP^R strains, each collected from the same patient before and after DAP treatment (Supplemental Table 1), and 32 VISA strains isolated from patients receiving VCM therapy (Supplemental Table 2). All bacteria were kept in a final concentration of 40% glycerol at -80°C. Unless otherwise stated, the bacterial glycerol stocks were revived through cultivation in MH broth (Becton Dickinson, USA) at 37°C with constant agitation.
Two methods of drug susceptibility tests were employed in this study: Etest for determining DAP and VCM MIC for all studied strains, and broth microdilution for determining sensitivity toward DAP and VCM. Etests were performed following the guidelines of the Clinical and Laboratory Standard Institute. Briefly, each bacterial culture with 0.5 McFarland turbidity was streaked onto an MH agar plate, and DAP and VCM Etest strips (bioMérieux, France) were placed on the bacterial lawn. The inhibition zone break points for each isolate were read after 48 h. For the broth microdilution method, the ranges of DAP (0.5, 1, 1.5, 1.75, 2, 2.25, 2.5, and 3 mg/L) and VCM (0.5, 1, 1.5, 2, 2.25, 2.5, 3 and 4 mg/L) were tested against each bacterial culture. According to their susceptibility profile, each strain was classified as single DAP- or VCM-resistance or cross-reduced susceptibility to DAP and VCM.

MgO nanoparticles with an average size of 20 nm were purchased from Nanostructured & Amorphous Materials, Inc. (Houston, TX, USA). ZnO nanoparticles (average size of 30 nm) were from Inframat Advanced Materials LLC (Manchester, CT, USA). A stock suspension (8 mg/ml) was freshly prepared by resuspending 80 mg of the nanoparticles into 10 ml ddH₂O for H₂O₂ production assay or Mueller Hinton broth (MH broth; Becton–Dickinson Co., Sparks, MD, USA) for cell culture experiments. All of the nanoparticle suspensions were homogenized by vigorous vortexing prior to use in the following experiments.