daptomycin  (Thermo Fisher)

Journal: Microorganisms

Article Title: Alanine Uptake Is Required to Maintain Staphylococcus aureus Cell Envelope Stability Under Magnesium and Calcium Limitation

doi: 10.3390/microorganisms14061332

Figure Lengend Snippet: Tolerance of S. aureus to membrane perturbation by Triton X-100 in low Mg 2+ and Ca 2+ or daptomycin treatment requires efficient alanine uptake or 2 mM Mg 2+ /Ca 2+ . ( A ) Lysis in the presence of 0.05% Triton X-100 of the wild-type JE2, the alanine transporter mutant ( aapA ::tn), or the complemented aapA mutant ( aapA ::tn pRMC2:: aapA ) in defined medium containing 0 alanine and low (0.04 mM each) Mg 2+ and Ca 2+ or with the addition of 1 mM L-alanine in low Mg 2+ and Ca 2+ , or 1 mM L-alanine and 2 mM Mg 2+ and Ca 2+ . ( B ) Growth of wild-type JE2 or alanine transporter mutant aapA ::tn in defined medium containing 2 mM Ca 2+ and 0.1 or 1 mM L-alanine with or without 1 µM Daptomycin. Data are displayed as the mean +/− SEM of 3 biological replicates containing 3 technical replicates per experiment.

Article Snippet: Susceptibility to daptomycin (MedChemExpress, Monmouth Junction, NJ, USA) was tested according to our standard growth assay described above.

Techniques: Membrane, Lysis, Mutagenesis

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Polygenic, cell-envelope adaptations drive high-frequency daptomycin resistance in Staphylococcus capitis NRCS-A from neonatal sepsis and NEC

doi: 10.1128/aac.01414-25

Figure Lengend Snippet: ( A ) Binding of BODIPY-labeled daptomycin (BODIPY-Dap) in the previously determined subinhibitory concentration of 7.81 µg/mL to the S. capitis NRCS-A isolate ScSK1 (light blue) and the non-invasive S. capitis DSM6717 (dark blue). Bacterial cells were incubated with BODIPY-Dap and analyzed by flow cytometry (FITC channel) at 0 h, 0.5 h, 1 h, and 2 h. Statistical analysis was performed using two-way ANOVA with Šidák’s multiple-comparisons test to compare BODIPY-Dap binding between strains at each time point ( P < 0.05, * P < 0.01, ** P < 0.001). Data are shown as mean ± SD from at least four independent experiments. ( B ) Fluorescence microscopy of S. capitis strains DSM6717 and ScSK1 after incubation with BODIPY-Dap for 0, 10, 30, and 60 min. Representative images were acquired on a Zeiss LSM 800 confocal microscope using a 100× objective. Scale bar, 2 µm.

Article Snippet: BODIPY 493/503 (4,4-Difluoro-1,3,5,7,8-Pentamethyl-4-Bora-3a,4a-Diaza-s-Indacen) (Invitrogen/Thermo-Fisher, D3922) labeling of daptomycin was performed as described previously ( , ).

Techniques: Binding Assay, Labeling, Concentration Assay, Incubation, Flow Cytometry, Fluorescence, Microscopy

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Polygenic, cell-envelope adaptations drive high-frequency daptomycin resistance in Staphylococcus capitis NRCS-A from neonatal sepsis and NEC

doi: 10.1128/aac.01414-25

Figure Lengend Snippet: Daptomycin and vancomycin susceptibility of S. capitis NRCS-A isolates (ScSK) from our local collection as determined by MIC test strip. MICs of ( A ) daptomycin and ( B ) vancomycin are shown. Black lines indicate cut-off concentrations for resistance: 1 μg/mL for daptomycin, 2 μg/mL for vancomycin reduced susceptibility (cut-off for S. aureus ), and 4 μg/mL for vancomycin resistance (EUCAST definition, v15.0 of 2025). The S. aureus SA113 WT and SA113 ∆ mprF (gray), which is more susceptible to daptomycin and vancomycin, were used as controls. NRCS-A isolates (light blue) were compared with non-invasive S. capitis strain DSM6717 (dark blue) and the S. capitis bloodstream isolate from a toddler’s catheter sepsis (ScSK11, turquoise). The mean ± SEM of at least three independent experiments is shown.

Article Snippet: Therefore, MIC test strips for daptomycin and vancomycin (Liofilchem) were applied on MHB agar plates and analyzed after 24 h incubation at 37°C according to the manufacturer’s protocol.

Techniques: Stripping Membranes

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Polygenic, cell-envelope adaptations drive high-frequency daptomycin resistance in Staphylococcus capitis NRCS-A from neonatal sepsis and NEC

doi: 10.1128/aac.01414-25

Figure Lengend Snippet: ( A ) Binding of BODIPY-labeled daptomycin (BODIPY-Dap) in the previously determined subinhibitory concentration of 7.81 µg/mL to the S. capitis NRCS-A isolate ScSK1 (light blue) and the non-invasive S. capitis DSM6717 (dark blue). Bacterial cells were incubated with BODIPY-Dap and analyzed by flow cytometry (FITC channel) at 0 h, 0.5 h, 1 h, and 2 h. Statistical analysis was performed using two-way ANOVA with Šidák’s multiple-comparisons test to compare BODIPY-Dap binding between strains at each time point ( P < 0.05, * P < 0.01, ** P < 0.001). Data are shown as mean ± SD from at least four independent experiments. ( B ) Fluorescence microscopy of S. capitis strains DSM6717 and ScSK1 after incubation with BODIPY-Dap for 0, 10, 30, and 60 min. Representative images were acquired on a Zeiss LSM 800 confocal microscope using a 100× objective. Scale bar, 2 µm.

Article Snippet: Therefore, MIC test strips for daptomycin and vancomycin (Liofilchem) were applied on MHB agar plates and analyzed after 24 h incubation at 37°C according to the manufacturer’s protocol.

Techniques: Binding Assay, Labeling, Concentration Assay, Incubation, Flow Cytometry, Fluorescence, Microscopy

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Polygenic, cell-envelope adaptations drive high-frequency daptomycin resistance in Staphylococcus capitis NRCS-A from neonatal sepsis and NEC

doi: 10.1128/aac.01414-25

Figure Lengend Snippet: DAP-R evolution of S. capitis NRCS-A (ScSK4) in light blue compared to non-invasive S. capitis (DSM6717) in dark blue, S. aureus (USA300) in gray, and S. epidermidis (SeSK1) in white. Strains were grown without ( A ) or with ( B ) daptomycin pressure or ( C ) vancomycin pressure in MHB for 24 h followed by daptomycin MIC determination after each passage via E-tests. Black lines indicate cut-off concentrations for daptomycin resistance at 1 μg/mL (EUCAST definition, v15.0 of 2025). The black arrows highlight the passaging days on which bacterial strains evolved a DAP-R phenotype.

Article Snippet: Therefore, MIC test strips for daptomycin and vancomycin (Liofilchem) were applied on MHB agar plates and analyzed after 24 h incubation at 37°C according to the manufacturer’s protocol.

Techniques: Passaging