Colistin

Aerobic culture swabs were plated on Columbia blood agar, Colistin, Naladixic Blood Agar (CNA), and MaConkey Agar (Remel, Thermo Fisher Scientific, Waltham, MA, USA) both at 37°C with 5% CO₂, and at 25°C in room air. Anaerobic culture swabs were plated on Brucella Agar, Laked blood w/ kanamycin, vancomycin (LKV Agar), and phenol ethyl alcohol agar (PEA) (Remel, Thermo Fisher Scientific, Waltham, MA, USA) at 37°C in anaerobic gas mixture. Organisms were identified using gram negative identification plate (GNID) and/or gram positive identification plate (GPID) panels (VersaTREK and Sensititre, Thermo Fisher Scientific, Waltham, MA, USA). Fastidious and/or non-fermenting gram negative organisms were identified using GN2 microplate panels (Biolog, Hayward CA, USA). Anaerobes were identified by the Wadsworth Disk method [36 ] or anaerobic identification test panel microplates (Biolog, Hayward CA, USA).

A loopful of each stool sample was inoculated in 2 mL Lysogeny broth and incubated aerobically for 6 h at 37 °C to promote bacterial growth (Marincola et al., 2021 (link)). The broth was then sub-cultured onto 5% sheep blood agar plates supplemented with 10 μg/mL each of nalidixic acid and colistin (Thermofisher, City, United States) to suppress the growth of Gram-negative bacteria. Isolates were selected based on colony morphology consistent with staphylococci. A maximum of five morphologically similar or 10 morphologically dissimilar colonies per sample were randomly selected. This approach enabled us to describe the distribution of different strains colonizing an individual. Staphylococcal isolates were further confirmed using catalase and bile esculin agar (both from NHLS Media Laboratory, Greenpoint, South Africa). Isolates that were bile esculin negative but catalase positive represent staphylococci. The isolates were speciated using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS; VITEK® MS, BioMérieux, France).

Antibiotic susceptibility was determined by disk diffusion method. The tested antibiotics were cefoxitin, ceftriaxone, ertapenem, imipenem, meropenem, gentamicin, amikacin, nalidixic acid, ciprofloxacin, tetracycline, erythromycin, piperacillin/tazobactam, cefoperazone/sulbactam and colistin (Thermo Fisher Scientific, USA). colistin resistance was further confirmed by the broth microdilution method. The minimum inhibitory concentrations (MICs) of colistin were determined by the broth microdilution in cation-adjusted Mueller–Hinton II broth according to Clinical and Laboratory Standards Institute (CLSI) 2017 guidelines [16 ]. E. coli ATCC 25922 was used as a control and a range of colistin dilutions (Chem-Impex Int’l Inc., USA) between 0.25 mg/L and 128 mg/L were performed. Breakpoints of colistin susceptibility defined by European Committee on Antimicrobial Susceptibility Testing (EUCAST) [17 ] were used as follows: isolates with a colistin MIC ≤ 2 mg/L were categorised as susceptible, and those with a colistin MIC > 2 mg/L were categorised as resistant.

Mid-log phase cultures of A. baumannii were diluted to OD₆₀₀ 0.5 in 5 mM HEPES (pH 7.2) and 100 µL was transferred to clear-bottomed 96-well microtiter plates (Corning). Kaempferol was added to the final concentration of 0.375 mM and colistin sulphate (in 5 mM HEPES, Thermo Scientific) was added to a final concentration of 1.22 µg/mL (for experimental conditions) or 16 µg/mL (for positive control). Equal volumes of the kaempferol carrier (DMSO) was included in colistin only wells as required. 1-N-phenylnaphthylamine (NPN) (Acros Organics) was then added to a final concentration of 10 μM. Immediately after the addition of NPN, fluorescence was measured at 1-minute intervals for 20 min using a Synergy H1 microplate reader (BioTek); the excitation wavelength was set to 355 nm and emission was recorded at 405 nm^{80 (link)}.

The antibiotics ampicillin, tetracycline, ciprofloxacin, aztreonam, and colistin were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Polymyxin B was purchased from Research Products International (RPI), and gentamicin from VWR Life Science. The human AMP LL-37 was obtained from Peptide Sciences (Henderson, NV, USA). Stock solutions (1 or 2 mg/mL) of all antimicrobials were made by dissolving the powders in sterile distilled water, except for tetracycline which was dissolved in 70% ethanol. Solutions were stored at either 4 °C or −20 °C (according to safety and handling guidelines), and dilutions were prepared on the day of use.

The antibiotics (AB) used in this study were amoxicillin, ampicillin, doxycycline, lincomycin, neomycin, penicillin G, and colistin (all obtained from Alpha Aesar, Thermo Fisher GmbH, Kandel, Germany); AB stock solutions were prepared in BHI. Organic acids (OA) and nature-identical compounds (NIC) utilized in this study were citric acid, sorbic acid, benzoic acid, butyric acid, hexanoic acid, formic acid, fumaric acid, lactic acid, malic acid, and propionic acid (stocks prepared in BHI); and octanoic acid, decanoic acid, dodecanoic acid, thymol, carvacrol, eugenol, vanillin, α-pinene, eucalyptol, limonene, linalool, and menthol (stocks prepared in BHI supplemented with ethanol at a final concentration ≤ 3.5% to increase solubility); all OA and NIC were obtained from Merck KGaA, Darmstadt, Germany. Each solution was buffered to ensure a final pH of 6.5, filter-sterilized and diluted in sterile BHI to reach the final concentration tested.