Colistin

Stool samples were cultured on a MacConkey agar (Sigma-Aldrich, South Africa) containing 10 milligrams per litre (mg/L) vancomycin (Sigma-Aldrich) and 2 mg/L colistin (Sigma-Aldrich) to isolate colistin-resistant E. coli and K. pneumoniae using the spread plate method. Distinct bacterial colonies were selected based on their morphological resemblance to E. coli or Klebsiella spp. on the MacConkey agar. Isolates were sub-cultured on a chromogenic UriSelect agar (NHLS Media Laboratory, Green Point, South Africa) for preliminary identification, and matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF; Bruker Daltonics, Bremen, Germany) was performed to confirm species identification.
colistin resistance was confirmed by broth microdilution (BMD) following the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines and clinical breakpoints version 10 (minimum inhibitory concentration [MIC] ≤ 2 mg/L: susceptible, MIC > 2 mg/L: resistant). Escherichia coli American Type Culture Collection (ATCC) 25922 and E. coli National Collection of Type Cultures (NCTC) 13846 (mcr-1 positive) were used as colistin-susceptible and -resistant control strains, respectively.

Feces and large-intestine scraping were cultivated with selective medium on TSA Tryptic Soy Agar (Tryptic Soy Agar, DIFCO, Taiwan, China, cat no. 211043) supplemented with 5% sheep blood, 6.25 mg/μL rifampicin (rifampicin, Sigma-Aldrich, St. Louis, MO, USA, cat no. R3501), and 800 mg/L spectinomycin (Sigma-Aldrich, cat no. S9007), 25 mg/μL vancomycin (vancomycin, Sigma-Aldrich, cat no. V2002), 25 mg/L of colistin (colistin, Sigma-Aldrich, cat no. C1511) [32 (link)], and incubated for at least three days at 42 °C in jars with an anaerobic atmosphere. Anaerobic conditions were generated using a vacuum pump filled with a mixture of N₂ (80%), CO₂ (10%), and H₂ (10%) gases. To obtain pure colonies, several passages were performed until isolation. The isolates were stored in a freezer at −80 °C.
For differential diagnosis, at the end of the experiment, mucosal scrapings from the small intestine were cultivated on blood agar and MacConkey to evaluate the growth of enterotoxigenic Escherichia coli and scrapings from the large intestine mucosa were cultivated in Rappaport broth and Hectoein agar for Salmonella spp.

The LPS-deficient A. baumannii was isolated as described.^{49 (link)} In brief, an OD₆₀₀ of 1.0 of A. baumannii Ab3 was plated on LB agar containing 10 μg/mL of colistin (Sigma-Aldrich). Isolated colonies were picked and replica plated on LB agar containing vancomycin (Sigma-Aldrich, 10 μg/mL) and LB agar containing colistin (10 μg/mL). Colonies sensitive to vancomycin, but resistant to colistin were deemed LPS-deficient.

MIC of colistin (Sigma, Spain), MIC of niclosamide (Sigma, Spain), and MIC of colistin in presence of different concentrations of niclosamide (between 0.5 and 4 μM) against Col-S and Col-R references and clinical A. baumannii and K. pneumoniae strains were determined in two independent experiments by broth microdilution assay according to CLSI recommendations for A. baumannii and EUCAST recommendations for K. pneumoniae (Clinical and Laboratory Standards Institute, 2016 European Committee on Antimicrobial Susceptibility European Committee on Antimicrobial Susceptibility Testing [EUCAST], 2016 ). The initial inoculum of 5 x 10⁵ CFU/mL for each strain was used in microtiter plates V (Deltlab, Spain) in presence of colistin, niclosamide, or colistin plus niclosamide, and incubated for 16–18 h at 37°C. Escherichia coli ATCC 25922 was used as control strain.

As spectrofluorimetric and spectrophotometric binding studies were not informative of colistin binding to HSA due to the lack of optical variations following the interaction, we performed an indirect assay. In particular, colistin binding to HSA was evaluated by measuring the MG1655 E. coli strain (ATCC® 47046; Manassas, VA, USA) growth. Briefly, the activity of colistin (Sigma-Aldrich, St. Louis, MO, USA) and HSA (Sigma-Aldrich) on the MG1655 E. coli strain was tested in 96-well microtiter plates. In order to obtain high cell densities, bacterial cells were grown overnight in Mueller Hinton Broth 2 (MH II) (Sigma-Aldrich) and then diluted to an OD600 of 0.001 in MH II broth containing increasing concentrations of colistin (1, 1.25, and 1.5 μg/mL) and HSA (25, 50, 100, and 200 μg/mL). Microtiter plates were incubated for 20 h at 37 °C. Bacteria growth was measured at a wavelength of 600 nm using a microplate reader (Spark, Tecan, Switzerland). Biochemical results are shown as the means ± standard deviation (SD) derived minimally from three independent experiments. Differences between means, assessed by the Student’s t-test (GraphPad InStat 3.1 Software Inc., San Diego, CA, USA), were considered significant when p values were ≤0.05.

The disc diffusion test [25 (link)] was used for testing the susceptibility of K. pneumoniae isolates to a range of antimicrobials approved for both human and livestock use [26 ], including ampicillin (10 µg), amoxicillin-clavulanic acid (30 µg), ceftazidime (30 µg), cefepime (30 µg), amikacin (30 µg), nalidixic acid (30 µg), ciprofloxacin (5 µg), imipenem (10 µg), azithromycin (15 µg), aztreonam (30 µg), gentamicin (10 µg), tetracycline (30 µg), chloramphenicol (30 µg), nitrofurantoin (300 µg), trimethoprim-sulfamethoxazole (25 µg) and colistin (25 µg).
The broth microdilution method [27 ] was used for the determination of the minimum inhibitory concentrations (MICs) of colistin (Sigma-Aldrich, Seelze, Germany). A K. pneumoniae ATCC 700603 reference strain was included as quality control in the disc diffusion test and the fully colistin-susceptible E. coli ATCC 25922 and the mcr-1-positive E. coli NCTC 13846 with a colistin MIC of 4 mg/L were used in broth microdilution method.The results of antimicrobial susceptibility testing were interpreted according to the Clinical and Laboratory Standards Institute and European Committee on Antimicrobial Susceptibility Testing (EUCAST) joint subcommittee [28 ]. The multiple antibiotic resistance (MAR) index for each isolate was calculated according to Tambekaret al. [29 (link)].