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The importance of growth kinetic analysis in determining bacterial susceptibility against antibiotics and silver nanoparticles.

Theophel K, Schacht VJ, Schlüter M, Schnell S, Stingu CS, Schaumann R, Bunge M - Front Microbiol (2014)

Bottom Line: Despite the delayed onset of growth, treatment with fosfomycin, daptomycin, fusidic acid, cefoxitin, or gentamicin resulted in higher maximum growth rates and/or higher final optical density values compared with antibiotic-free controls, indicating that growth stimulation and hormetic effects may occur with extended exposure to sublethal antibiotic concentrations.Whereas neither maximum growth rate nor final cell density correlated with antibiotic concentration, the lag phase duration for some antibiotics was a more meaningful indicator of dose-dependent growth inhibition.Our results also reveal that non-temporal growth profiles are only of limited value for cultivation-based antimicrobial silver nanoparticle susceptibility testing.

View Article: PubMed Central - PubMed

Affiliation: Institute of Applied Microbiology, Research Center for BioSystems, Land Use, and Nutrition, Justus Liebig University of Giessen Giessen, Germany.

ABSTRACT
Routine antibiotics susceptibility testing still relies on standardized cultivation-based analyses, including measurement of inhibition zones in conventional agar diffusion tests and endpoint turbidity-based measurements. Here, we demonstrate that common off-line monitoring and endpoint determination after 18-24 h could be insufficient for reliable growth-dependent evaluation of antibiotic susceptibility. Different minimal inhibitory concentrations were obtained in 20- and 48 h microdilution plate tests using an Enterococcus faecium clinical isolate (strain UKI-MB07) as a model organism. Hence, we used an on-line kinetic assay for simultaneous cultivation and time-resolved growth analysis in a 96-well format instead of off-line susceptibility testing. Growth of the Enterococcus test organism was delayed up to 30 h in the presence of 0.25 μg mL(-1) of vancomycin and 8 μg mL(-1) of fosfomycin, after which pronounced growth was observed. Despite the delayed onset of growth, treatment with fosfomycin, daptomycin, fusidic acid, cefoxitin, or gentamicin resulted in higher maximum growth rates and/or higher final optical density values compared with antibiotic-free controls, indicating that growth stimulation and hormetic effects may occur with extended exposure to sublethal antibiotic concentrations. Whereas neither maximum growth rate nor final cell density correlated with antibiotic concentration, the lag phase duration for some antibiotics was a more meaningful indicator of dose-dependent growth inhibition. Our results also reveal that non-temporal growth profiles are only of limited value for cultivation-based antimicrobial silver nanoparticle susceptibility testing. The exposure to Ag(0) nanoparticles led to plasma membrane damage in a concentration-dependent manner and induced oxidative stress in Enterococcus faecium UKI-MB07, as shown by intracellular ROS accumulation.

No MeSH data available.


Related in: MedlinePlus

Silver-nanoparticle mediated ROS accumulation in Enterococcus cells, shown by green fluorescence signals after carboxy-H2DCFDA staining, identical sections after counterstaining with Hoechst 33342 (A), and assessment of cell membrane integrity at different Ag(0) nanoparticle concentrations (B). Representative images in (B) show SYTO 9 stained (green) Enterococcus cells with intact cell membranes and propidium iodide (red) stained cells with compromised cell membranes. SEM-EDX image of Ag(0) nanoparticles (0.006%, w/v) attached to cell debris in Enterococcus sp. cultures (C).
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Figure 3: Silver-nanoparticle mediated ROS accumulation in Enterococcus cells, shown by green fluorescence signals after carboxy-H2DCFDA staining, identical sections after counterstaining with Hoechst 33342 (A), and assessment of cell membrane integrity at different Ag(0) nanoparticle concentrations (B). Representative images in (B) show SYTO 9 stained (green) Enterococcus cells with intact cell membranes and propidium iodide (red) stained cells with compromised cell membranes. SEM-EDX image of Ag(0) nanoparticles (0.006%, w/v) attached to cell debris in Enterococcus sp. cultures (C).

Mentions: The accumulation of intracellular ROS was assessed with a carboxy-H2DCFDA-based assay and accompanying examination by fluorescence microscopy. The exposure to Ag(0) nanoparticles led to intracellular accumulation of ROS as shown by strong green fluorescence in samples derived from 0.006% Ag(0) nanoparticle treatments (Figure 3A, right panels). Accumulation of ROS in Enterococcus cells did occur to a much lesser extent in the absence of Ag(0), as indicated by the presence of only weak fluorescence signals in Ag(0)-free control incubations (Figure 3A, left panels).


The importance of growth kinetic analysis in determining bacterial susceptibility against antibiotics and silver nanoparticles.

Theophel K, Schacht VJ, Schlüter M, Schnell S, Stingu CS, Schaumann R, Bunge M - Front Microbiol (2014)

Silver-nanoparticle mediated ROS accumulation in Enterococcus cells, shown by green fluorescence signals after carboxy-H2DCFDA staining, identical sections after counterstaining with Hoechst 33342 (A), and assessment of cell membrane integrity at different Ag(0) nanoparticle concentrations (B). Representative images in (B) show SYTO 9 stained (green) Enterococcus cells with intact cell membranes and propidium iodide (red) stained cells with compromised cell membranes. SEM-EDX image of Ag(0) nanoparticles (0.006%, w/v) attached to cell debris in Enterococcus sp. cultures (C).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4226228&req=5

Figure 3: Silver-nanoparticle mediated ROS accumulation in Enterococcus cells, shown by green fluorescence signals after carboxy-H2DCFDA staining, identical sections after counterstaining with Hoechst 33342 (A), and assessment of cell membrane integrity at different Ag(0) nanoparticle concentrations (B). Representative images in (B) show SYTO 9 stained (green) Enterococcus cells with intact cell membranes and propidium iodide (red) stained cells with compromised cell membranes. SEM-EDX image of Ag(0) nanoparticles (0.006%, w/v) attached to cell debris in Enterococcus sp. cultures (C).
Mentions: The accumulation of intracellular ROS was assessed with a carboxy-H2DCFDA-based assay and accompanying examination by fluorescence microscopy. The exposure to Ag(0) nanoparticles led to intracellular accumulation of ROS as shown by strong green fluorescence in samples derived from 0.006% Ag(0) nanoparticle treatments (Figure 3A, right panels). Accumulation of ROS in Enterococcus cells did occur to a much lesser extent in the absence of Ag(0), as indicated by the presence of only weak fluorescence signals in Ag(0)-free control incubations (Figure 3A, left panels).

Bottom Line: Despite the delayed onset of growth, treatment with fosfomycin, daptomycin, fusidic acid, cefoxitin, or gentamicin resulted in higher maximum growth rates and/or higher final optical density values compared with antibiotic-free controls, indicating that growth stimulation and hormetic effects may occur with extended exposure to sublethal antibiotic concentrations.Whereas neither maximum growth rate nor final cell density correlated with antibiotic concentration, the lag phase duration for some antibiotics was a more meaningful indicator of dose-dependent growth inhibition.Our results also reveal that non-temporal growth profiles are only of limited value for cultivation-based antimicrobial silver nanoparticle susceptibility testing.

View Article: PubMed Central - PubMed

Affiliation: Institute of Applied Microbiology, Research Center for BioSystems, Land Use, and Nutrition, Justus Liebig University of Giessen Giessen, Germany.

ABSTRACT
Routine antibiotics susceptibility testing still relies on standardized cultivation-based analyses, including measurement of inhibition zones in conventional agar diffusion tests and endpoint turbidity-based measurements. Here, we demonstrate that common off-line monitoring and endpoint determination after 18-24 h could be insufficient for reliable growth-dependent evaluation of antibiotic susceptibility. Different minimal inhibitory concentrations were obtained in 20- and 48 h microdilution plate tests using an Enterococcus faecium clinical isolate (strain UKI-MB07) as a model organism. Hence, we used an on-line kinetic assay for simultaneous cultivation and time-resolved growth analysis in a 96-well format instead of off-line susceptibility testing. Growth of the Enterococcus test organism was delayed up to 30 h in the presence of 0.25 μg mL(-1) of vancomycin and 8 μg mL(-1) of fosfomycin, after which pronounced growth was observed. Despite the delayed onset of growth, treatment with fosfomycin, daptomycin, fusidic acid, cefoxitin, or gentamicin resulted in higher maximum growth rates and/or higher final optical density values compared with antibiotic-free controls, indicating that growth stimulation and hormetic effects may occur with extended exposure to sublethal antibiotic concentrations. Whereas neither maximum growth rate nor final cell density correlated with antibiotic concentration, the lag phase duration for some antibiotics was a more meaningful indicator of dose-dependent growth inhibition. Our results also reveal that non-temporal growth profiles are only of limited value for cultivation-based antimicrobial silver nanoparticle susceptibility testing. The exposure to Ag(0) nanoparticles led to plasma membrane damage in a concentration-dependent manner and induced oxidative stress in Enterococcus faecium UKI-MB07, as shown by intracellular ROS accumulation.

No MeSH data available.


Related in: MedlinePlus