Susceptibility testing was performed for each included strain in a broth microdilution after EUCAST (European Committee on Antimicrobial Susceptibility Testing) guidelines, determining the MIC of gepotidacin towards each strain. Additionally, MICs of 100 strains towards levofloxacin and moxifloxacin were assessed in a broth microdilution. The anti-infective agents were diluted in solvent (dimethyl sulfoxide, DMSO) and Mueller Hinton Bouillon 2 for the preparation of stock solutions. Working solutions were prepared by dilution in Mueller Hinton Bouillon 2 and all solutions were stored at −20°C. Broth microdilution with gepotidacin, levofloxacin and moxifloxacin (MedChemExpress LLC, Monmouth, NJ, USA) was performed in concentrations between 0.03 and 16 mg/L. Additionally, MIC strip tests (Liofilchem srl, Roseto degli Abruzzi, Italy) were performed for co-trimoxazole for a total of 100 strains.
Mic strip tests
Manufactured by Liofilchem
Sourced in Italy
MIC strip tests are a laboratory tool used to determine the minimum inhibitory concentration (MIC) of antimicrobial agents against bacterial isolates. The tests consist of paper strips coated with a gradient of antibiotic concentrations, which are applied to inoculated agar plates to establish the lowest concentration of the antimicrobial agent that inhibits the growth of the tested microorganism.
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3 protocols using mic strip tests
1
Susceptibility Testing of Novel Antibiotics
2
Retrospective AMR Profile Analysis
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AMR profiles of isolates associated with the retrospectively analysed spectra were accessed through the laboratory information systems of the USB and the LTW (n = 7876). The accessed AMR profiles were measured in clinical routine diagnostics from January 2015 to June 2018 using either microdilution methods (Vitek2, AST-N242 GN Cards, bioMérieux), MIC strip tests (Liofilchem, Roseto degli Abruzzi, Italy) or disc diffusion tests (ThermoFisher Scientific, Waltham, USA). Breakpoints were interpreted as susceptible or resistant according to the current EUCAST Breakpoint table (v6.0 – 8.1) [50 ].
3
Vancomycin-Resistant Enterococcus Characterization
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The Department of Medical Microbiology, Semmelweis University, Budapest, Hungary cultured and characterized 44 VRE strains. All the strains were isolated from routine laboratory samples of patients suffering from wound, urinary tract or blood stream infections. Clinical samples were cultured on COS agar (Columbia agar+5% sheep blood, Biomérieux, Budapest, Hungary), and vancomycin-resistant strains were isolated on VRE chromogenic medium (Biomérieux). Species of strains were identified by MALDI-TOF-MS. Minimum inhibitory concentrations (MICs; mg l -1 ) were determined for vancomycin and teicoplanin on Mueller Hinton E agar (Biomérieux using MIC strip tests (Liofilchem, Roseto degli Abruzzi, Italy), according to the manufacturer's instructions, and by using a direct colony suspension equivalent to a McFarland standard of 0.5. Testing conditions also included incubation at 35.5 °C for 24 h. All results were interpreted by using breakpoints for susceptibility and resistance according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) 22 Concerning vancomycin/ teicoplanin MIC breakpoints, strains with an MIC44 or 2 mg l -1 were considered to be vancomycin-or teicoplanin-resistant, respectively.
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