The genomic location of the mcr-2 gene was verified by S1 nuclease digestion of genomic DNA followed by electrophoretic separation and Southern hybridization. A digoxigenin-labeled DNA probe (“DIG luminescent detection Kit”, Boehringer Mannheim GmbH, Mannheim) targeting a 567-bp PCR fragment specific for the mcr-2 gene using the aforementioned primers (Xavier et al., 2016 (link)) was used. In addition, we performed an in silico search of whole genome sequences with mlplasmids v. 1.0.0 (Arredondo-Alonso et al., 2018 (link)). To test whether the colistin resistance determinant was transferable, conjugation was performed by the broth filter mating method at 37°C using plasmid-free sodium azide resistant E. coli K12-J53 (J53 AziR) as recipient. Prior to the conjugation assays, all mcr-2-positive isolates were tested for their susceptibility to sodium azide. Transconjugants were selected on Endo agar plates containing 100 mg/L sodium azide and 2 mg/L colistin sulfate or containing 100 mg/L sodium azide and 4 mg/L colistin sulfate (Sigma-Aldrich, Germany, Karlsruhe, Germany). To confirm successful plasmid transfer, antimicrobial susceptibility testing of transconjugants, a PCR targeting the mcr-2 gene and plasmid profiling was performed as described above.
Colistin sulfate
Manufactured by Merck Group
Sourced in United States, Germany, France, United Kingdom, Italy, Australia, India
Colistin sulfate is a laboratory-grade chemical compound used in various research and testing applications. It is a white, crystalline powder that serves as a bactericidal agent, primarily targeting Gram-negative bacteria. Colistin sulfate is a common component in growth media and other microbiological applications.
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Lab products found in correlation
Ampicillin, by Merck Group (6 mentions)
Ciprofloxacin, by Merck Group (6 mentions)
Gentamicin sulfate, by Merck Group (4 mentions)
Vancomycin hydrochloride, by Merck Group (4 mentions)
Tigecycline, by Pfizer (4 mentions)
Ceftazidime, by Merck Group (4 mentions)
Gradient test strips, by Liofilchem (3 mentions)
Levofloxacin, by Merck Group (3 mentions)
Type 1 dextrin from corn, by Merck Group (3 mentions)
Tobramycin, by Merck Group (3 mentions)
Minocycline hydrochloride, by Merck Group (3 mentions)
Imipenem, by Merck Group (3 mentions)
Cls3640, by Corning (3 mentions)
Vancomycin hcl, by Merck Group (3 mentions)
Piperacillin, by Merck Group (3 mentions)
Meropenem, by Merck Group (3 mentions)
Colistin, by Merck Group (3 mentions)
Fm4 64, by Thermo Fisher Scientific (3 mentions)
Streptomycin sulfate, by Merck Group (2 mentions)
Clindamycin hydrochloride, by Merck Group (2 mentions)
91 protocols using colistin sulfate
1
Verification of mcr-2 Gene Location
2
Determining mcr-Harboring CRE Antibiotic Resistance
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Analysis of the minimal inhibitory concentration (MIC) of antimicrobials for mcr-harboring CRE isolates was performed using the Epsilometer test (E test) and interpreted according to the 2020 Clinical and Laboratory Standards Institute guidelines (Clinical and Laboratory Standards Institute, 2020 ), and E. coli ATCC 25922 was used as the control. The E test was based on ampicillin, amoxicillin-clavulanate, ampicillin-sulbactam, piperacillin-tazobactam, cefazolin, cefepime, cefotaxime, cefoxitin, ertapenem, meropenem, imipenem, gentamicin, amikacin, ciprofloxacin, levofloxacin, trimethoprim, fosfomycin, nitrofurantoin, chloramphenicol, tetracycline, aztreonam, and azithromycin. Additionally, a modified carbapenem inactivation method (mCIM) was applied to all CRE isolates according to 2020 CLSI M100-S30.
Broth microdilution was used to determine the MIC of colistin using colistin sulfate (Merck, Germany) at 1, 2, 4, 8, 16, and 32 μg/ml, respectively. According to 2020 CLSI M100-S30, an MIC of ≤2 μg/ml was interpreted as intermediate susceptibility, whereas an MIC of ≥4 μg/ml was considered to indicate resistance.
Broth microdilution was used to determine the MIC of colistin using colistin sulfate (Merck, Germany) at 1, 2, 4, 8, 16, and 32 μg/ml, respectively. According to 2020 CLSI M100-S30, an MIC of ≤2 μg/ml was interpreted as intermediate susceptibility, whereas an MIC of ≥4 μg/ml was considered to indicate resistance.
3
Formulation of Chitosan-Based Antimicrobial Agents
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P. aeruginosa ATCC® 27853 ™ was obtained from the American Type Culture Collection (ATCC; Rockville, MD, USA). Muller Hinton Cation-Adjusted Broth and Colistin Sulfate were purchased from Merck (Darmstadt, Germany). Cholesterol and dioleoyl phosphatidyl ethanolamine (DOPE) were obtained from Avanti Polar Lipids (Alabaster, AL, USA). Soy lecithin (Medick) was purchased from a local pharmacy (Cali-Colombia) and characterized in a recent study [21 (link)]. Chitosan with a deacetylation degree of >90% (MW = 477 KDa) was provided by the Laboratory of Design and Formulation of Chemicals and Derivatives of the Icesi University [22 (link)].
4
Antimicrobial Susceptibility Testing Protocol
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Antimicrobial susceptibility was determined using Kirby-Bauer disk diffusion method according to the clinical and laboratory standards institute (CLSI) guideline [11 ]. The minimal inhibitory concentration (MIC) of carbapenems (imipenem, meropenem and ertapenem) were determined by gradient test strips (Liofilchem, Italy). Broth microdilution method was utilized to determine the MIC of colistin using colistin sulfate (Merck, Germany). According to CLSI M100- S30, a MIC = 2 µg/ml was interpreted as intermediate susceptibility, whereas a MIC of ≥ 4 µg/ml was considered as resistance [11 ]. E. coli ATCC 25922 was used as a control strain for antimicrobial susceptibility testing.
5
Antibiotic Sensitivity of L. reuteri PSC102
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The antibiotic sensitivity of L. reuteri PSC102 was determined against 14 antibiotics, including cephalexin, colistin sulfate, enrofloxacin, cefalonium, amoxicillin trihydrate, penicillin G procaine, norfloxacin, spectinomycin, tylosin base, cefuroxime sodium, florfenicol, penicillin G benzathine, gentamicin sulfate, and streptomycin sulfate (Sigma-Aldrich, St. Louis, MO, USA), as previously described [26 (link)]. Briefly, 100 µL of cultured L. reuteri PSC102 was mixed with 100 µL of diluted antibiotic solution in a 96-well plate. After finally adjusting the concentration to 106 CFU/mL, the bacteria were incubated for 24 h at 37 °C. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined by measuring the OD value at 600 nm using Gen5 microplate reader version 3.08 (BioTek, Winooski, VT, USA) and by streaking on Mueller–Hinton agar plate, respectively.
6
Bioluminescent Bacterial Infection Assay
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Bacterial strains used in this work are listed in Table 1 . E. ictaluri 93–146 WT strain and vaccine strains were cultured in BHI agar or broth (Difco, Sparks, MD, United States) and incubated throughout the study at 30°C. Each vaccine strain was labeled with bioluminescence by transferring pAKgfplux1 from an E. coli donor strain (SM10aaapir) by conjugation as described previously (Karsi and Lawrence, 2007 (link)). When required, media were supplemented with following antibiotics and reagents; ampicillin (Amp: 100 mg/ml), and colistin sulfate (Col: 12.5 mg/ml, Sigma–Aldrich, St. Louis, MN, United States).
Overnight cultures of E. ictaluri and vaccine strains were grown and prepared as described above, followed by two PBS washes before opsonization with channel catfish serum for 30 min at room temperature. Opsonized bacteria were then used for antigen uptake and infection experiments.
Overnight cultures of E. ictaluri and vaccine strains were grown and prepared as described above, followed by two PBS washes before opsonization with channel catfish serum for 30 min at room temperature. Opsonized bacteria were then used for antigen uptake and infection experiments.
7
Adherence of A. baumannii to A549 Cells
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Adherence of A. baumannii strains to A549 cells (human type 2 pneumocytes) was determined as described previously [4 (link)], with minor modifications. In brief, ~ 105 A549 cells were infected with ~ 107 bacterial CFU and incubated for 60 min at 37 °C in 5 % CO2 (v/v) atmosphere. Non-adherent bacterial cells were removed by washing with PBS. Infected cells were lysed by the addition of 1 ml distilled water and serial 10-fold dilutions were plated on LB agar to determine the number of CFU of adherent bacteria. To determine adherent and invading bacteria, A549 cells were infected with A. baumannii strains as described above. The monolayers were then treated with 1 ml of fresh culture medium containing 5 mg/L of colistin sulfate (Sigma-Aldrich, Milan, Italy) for further 30 min, the shortest time point that resulted in the killing of all extracellular bacteria added to the monolayers. Afterwards, the cells were washed with PBS, harvested with trypsin, and lysed with sterile distilled water. Dilutions from harvested samples were inoculated on LB agar plates and bacterial colony counts were estimated after overnight incubation at 37 °C. Each experiment was performed in triplicate.
8
Microbial Growth and Susceptibility Assay
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Blood and CLED agar plates, phosphate buffered saline (PBS; 10 mM; pH 7.4), lysogeny broth (LB; pH 7.5), cation-adjusted Muller-Hinton Broth (CaMHB), and sterilized deionized water (WID; pH 7.0) were obtained from the Substrate Unit at Karolinska University Hospital, Stockholm, Sweden. RPMI-1640 was purchased from Invitrogen. Sodium polyanethole sulfonate (SPS) and colistin sulfate were purchased from Sigma-Aldrich. Stock solutions were prepared in PBS at 1000 μg/ml for colistin and 10 mg/ml in sterilized distilled water for SPS. LL-37 was purchased from Innovagen (Lund, Sweden) and dissolved in 0.1% trifluoroacetic acid (TFA; Fluka BioChemika), aliquoted and stored at −20°C until used. The E3 medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, and 0.33 mM MgSO4) was provided by the zebrafish core facility at Karolinska Institutet, Stockholm, Sweden.
9
Colistin Resistance in Klebsiella pneumoniae Isolates
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A total of 20 col-R KP clinical strains isolated from the same number of patients hospitalized in Imam Khomeini hospital, the largest medical center of the country with 1,400 beds were studied. The isolates were obtained between 2015 and 2017 through antimicrobial resistance surveillance study for detection of col-R isolates. MICs of colistin were determined by broth macrodilution method using colistin sulfate (Sigma–Aldrich) over a range of dilutions from 0.125 to 128 μg/ml according to instructions described by Wiegand et al. (2008) (link) and Clinical and Laboratory Standards Institute (2014) . Isolates characterized with MIC values greater than 2 μg/ml were categorized as resistant according to guidelines described by the European Committee on Antimicrobial Susceptibility Testing (EUCAST)1 . K. pneumoniae ATCC 700603 was used as a quality control for antimicrobial susceptibility testing.
10
Synthesis and Preparation of DEA-C3D Compounds
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DEA-C3D potassium salt and DEA-C3D free carboxylic acid were synthesized at the University of Wollongong, Australia, and stored at −80°C with desiccant. Details of synthesis for both DEA-C3D forms are described elsewhere.11 (link),12 (link) Stock solutions of DEA-C3D at 25.6 mM were prepared fresh in DMAO (Sigma–Aldrich) and diluted in medium immediately prior to use. Stock solutions of tobramycin and colistin sulfate (Sigma–Aldrich) were prepared in water at 20 mg/L and refrigerated or kept frozen until use.
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