A. baumannii strains 4106, 4112, 4119, 3941 and 3942 were obtained from Walter Reed Army Institute for Research (WRAIR). A. baumannii strain 17978mcr−1 and K. pneumoniae strains B9, A5, C3 and F2210291mcr−1 were obtained from Professor Robert Ernst at The University of Maryland, Baltimore. Stock cultures were stored in 25% glycerol and maintained at −80 °C. Prior to use, colonies were grown on LB (Lennox) agar. CAMHB was purchased from BD Diagnostics. Colistin sulfate salt was purchased from Sigma Aldrich (Cat# C4461). All assays were run in duplicate and repeated at least two separate times.
Colistin sulfate salt
Manufactured by Merck Group
Sourced in United States, Germany, France
Colistin sulfate salt is a type of laboratory equipment used for various research and analytical applications. It serves as a salt form of the antimicrobial substance colistin, which is derived from the bacterium Paenibacillus polymyxa. This product is commonly utilized in microbiology and biochemistry research settings.
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96 well polystyrene microplate, by Corning (1 mentions)
Kanamycin, by Merck Group (1 mentions)
Phosphate buffer saline pbs, by Merck Group (1 mentions)
25 protocols using colistin sulfate salt
1
Antimicrobial Susceptibility Testing Protocol
2
Colistin Resistance Testing Protocol
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The lyophilized powder of colistin sulfate salt was purchased from Sigma-Aldrich (Merck, Germany) and was re-suspended in distilled sterile water. A final concentration of colistin vials (1,024 μg/ml) was stored at − 80 °C for further tests. In addition, Mueller–Hinton broth was prepared in separate tubes for different concentrations of colistin, ranging from 0.5 to 16 mg/L with two-fold dilutions according to the recommendations by CLSI/EUCAST guidelines. The E. coli isolates with MIC ≥ 4 mg/mL of colistin were determined as resistant. For each isolate tested, a positive and negative control were included in the first and second wells of the plate, respectively. E. coli ATCC 25,922 was used as a standard control [16 (link)].
3
Antibiotics Preparation and Stock Solutions
4
Colistin Susceptibility Testing Protocol
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MICs were determined by broth dilution method using cationic adjusted Mueller-Hinton broth and conducted following methods published by the CLSI (19 ). MICs ranged from 0.125 μg/mL to 128 μg/mL. Broth dilutions were replicated 10 times per isolate using separate isolated colonies for each replicate. If there were discordant MICs, broth dilutions were repeated in triplicate. The final MIC was the MIC of the concordant repeated triplicates. Colistin sulfate salt was acquired from Sigma-Aldrich (catalog number C4461; St. Louis, MO). Determination of intermediate and resistant organisms was done using the previously mentioned CLSI breakpoints.
5
Colistin Susceptibility Assessment by BMD
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Colistin-susceptibility and MIC in isolates were assessed by the ISO-20776 standard broth microdilution technique (BMD) jointly recommended by the EUCAST (2016a) with few modifications. In brief, a two-fold dilution (0.125–128 μg/mL) of the colistin sulfate salt (Sigma-Aldrich) was prepared on a 96 well microtitre plate. The bacterial inoculum was inoculated in each well with a final concentration of 5 × 105 CFU/mL. Then the microtitre plate was incubated at 37°C for 16–20 h. After the incubation period, 30 μL of 0.015% resazurin solution was added to each well of the microtitre plate and incubated again at 37C for 1 h, during which the plate was routinely checked every 15 min. The colistin MICs were interpreted with the naked eye and recorded by observing the color change of resazurin (discoloration from blue to pink or purple indicates colistin resistance, while susceptibility is deduced when no color change- blue color). The test was performed in triplicates. During colistin-susceptibility testing, colistin-susceptible E. coli ATCC25922 and colistin-resistant (ColR) E. coli NCTC 13846 were utilized as negative and positive controls, respectively.
6
Antibiotic Susceptibility Profiling of B. cepacia and E. cloacae
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Single colonies of B. cepacia complex isolate JC8 and E. cloacae Mu208 were inoculated into 1.5 mL Mueller–Hinton broth (BD, Product #275730), and cultures were grown overnight at 37 °C with shaking. Cultures were serially diluted in phosphate-buffered saline, and 10 µL of each dilution was spotted on Mueller–Hinton agar (BD, Product #225250) plates containing 0, 0·125, 0·25, 0·5, 1, 2, and 4 times the breakpoint concentration of each antibiotic. Antibiotics used were ticarcillin disodium (BioVision, Product #B1536) with clavulanate potassium salt (Cayman Chemical Company, Procut #19456), amikacin sulfate (AstaTech, Product # 40003), colistin sulfate salt (Sigma-Aldrich, Product # C4461), and fosfomycin disodium salt (TCI America, Product # F0889). For Mu208 on fosfomycin, broth and agar included 25 µg/mL glucose-6-phosphate (Sigma-Aldrich, Product #10127647001). Plates were maintained at 37 °C overnight for Mu208 and for 36 to 60 h for JC8.
7
Modified Porcine Surfactant Supplementation
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Poractant alpha (SF, Curosurf®, Chiesi Farmaceutici, Parma, Italy) is a modified porcine SF at a phospholipid concentration of 80 mg/mL. PxE sulfate (Colistin sulfate salt, Sigma Aldrich; No. C4461) or PxB sulfate (Sigma Aldrich No. P1004) were suspended in saline at 20 (PxE) or 80 mg/mL (PxB or PxE) and mixed with SF at a ratio of 1:100. The resulting phospholipid concentrations were 79.2 mg/mL in the PxB- or PxE-containing preparations which is close to the original SF preparation of 80 mg/mL. The polymyxin concentrations were 0.2 or 0.8 mg/mL.
8
Colistin Susceptibility Testing of Clinical Isolates
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In total, 60 GN clinical isolates obtained from Severance hospital, including P. aeruginosa (N=27) and ACB (N=33), were tested (Table 1 ). The study was approved by the Institutional Review Board of Yonsei University Health system, Seoul, Korea. (1-2017-0079). The clinical strains were collected from sputum and urine in 2017 and stored at −70℃.
Quality control was performed using E. coli (ATCC 25922), P. aeruginosa (ATCC 27853), and mcr-1-harboring K. aerogenes (a clinical isolate). Colistin MICs were determined by BMD for all 60 strains using colistin sulfate salt (Sigma-Aldrich, St. Louis, MO, USA) and polystyrene 96-well microplates (Corning, NY, USA). As shown inTable 1 , disk diffusion AST results were interpreted based on the breakpoints, which showed good agreement with the MIC determined by BMD according to the 2018 CLSI guidelines [21 ]. Each strain was tested using predetermined media, i.e., commercial MHA (100% agar concentration), MHA with 30% agar (MHA30), and MHA30 with 100 µg/mL protamine (MHA30P100), which were selected based on the optimization in phase I. The colistin disk diffusion test was performed using a 10 mg colistin disk on MHA plates that were incubated at 35℃ for 16–18 hours in 5% CO2.
Quality control was performed using E. coli (ATCC 25922), P. aeruginosa (ATCC 27853), and mcr-1-harboring K. aerogenes (a clinical isolate). Colistin MICs were determined by BMD for all 60 strains using colistin sulfate salt (Sigma-Aldrich, St. Louis, MO, USA) and polystyrene 96-well microplates (Corning, NY, USA). As shown in
9
Antimicrobial Peptide Synthesis and Characterization
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Colistin sulfate salt,
ampicillin, kanamycin, and phosphate saline
buffer (PBS) were purchased from Sigma-Aldrich, France. Bovine catestatin
has an amino acid sequence of RSMRLSFRARGYGFRGPGLQL, and it was purchased
from ProteoGenix (Schiltigheim, France). The peptides were stored
at −20 °C as a stock solution at 1 g/L in nonpyrogenic
sterile water (Aqua B-Braun, Melsungen, Germany). Structures and physicochemical
properties of colistin and catestin are reported inTable 1 .
ampicillin, kanamycin, and phosphate saline
buffer (PBS) were purchased from Sigma-Aldrich, France. Bovine catestatin
has an amino acid sequence of RSMRLSFRARGYGFRGPGLQL, and it was purchased
from ProteoGenix (Schiltigheim, France). The peptides were stored
at −20 °C as a stock solution at 1 g/L in nonpyrogenic
sterile water (Aqua B-Braun, Melsungen, Germany). Structures and physicochemical
properties of colistin and catestin are reported in
10
In vitro and in vivo antimicrobial evaluation
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For the in vitro assays, antimicrobials, colistin sulfate salt and rifampin, were used as standard laboratory powders (Sigma-Aldrich, Madrid, Spain). For in vivo experiments, clinical formulations were used: colistimethate sodium (CMS) (Genéricos Españoles S.A., Madrid, Spain) and rifampin (Sanofi-Aventis, Madrid, Spain).
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