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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

Effect of selected antibiotics on the growth dynamics of Enterococcus faecium strain UKI-MB07. Note the extended lag phase and partial growth promotion at low concentrations for some antibiotics. This hormetic effect was associated with higher maximum specific growth rates and increased OD values. Each data point (time resolution over 48 h: 15 min) represents mean values of triplicate cultivations, normalized with data from identical incubations in the absence of bacterial cells (sterile controls). Hence, each curve represents 576 single data points and kinetics from about 21,900 single data points are shown in this figure. For visual clarity, standard deviations (error bars) are only presented for selected time points.
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Figure 1: Effect of selected antibiotics on the growth dynamics of Enterococcus faecium strain UKI-MB07. Note the extended lag phase and partial growth promotion at low concentrations for some antibiotics. This hormetic effect was associated with higher maximum specific growth rates and increased OD values. Each data point (time resolution over 48 h: 15 min) represents mean values of triplicate cultivations, normalized with data from identical incubations in the absence of bacterial cells (sterile controls). Hence, each curve represents 576 single data points and kinetics from about 21,900 single data points are shown in this figure. For visual clarity, standard deviations (error bars) are only presented for selected time points.

Mentions: The effects of various antibiotics on the growth dynamics of the Enterococcus clinical isolate were monitored using an automated 96-well microtiter plate assay that allowed simultaneous cultivation and on-line analysis of bacterial growth. By examining growth over time using the automated method, different effective exposure times and concentration-dependent effects on the growth dynamics of the Enterococcus clinical isolate became evident. Despite the fact that categorization of data after 48 h of incubation remains “theoretical” since we did not follow the guideline incubation time for utilizing MIC interpretative standards for Enterococcus spp. (dilution methods: 16 to 20 h, 24 h for vancomycin; Clinical and Laboratory Standards Institute [CLSI], 2012, 2013), for antibiotics tested at two concentrations, the Enterococcus isolate was sensitive to cefotaxim (2/8), doxycyclin (1/4), cotrimoxazol (16/64), mupirocin (4/8), and erythromycin (1/4) in both the 20- and 48 h experiments. According to the current breakpoint interpretation (Clinical and Laboratory Standards Institute [CLSI], 2013), these data indicate that the Enterococcus test isolate can be categorized as susceptible (S) to doxycyclin and as either (S) or of intermediate susceptibility (I) to erythromycin (“theoretical” categorization of 48-h data sets). Ciprofloxacin showed intermediate performance (growth at 1 μg mL-1 and no growth at 2 μg mL-1) in both the 20- and 48-h experiments; however, growth in the presence of 1 μg mL-1 of ciprofloxacin did not begin until about 15 h of incubation (Figure 1A).


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)

Effect of selected antibiotics on the growth dynamics of Enterococcus faecium strain UKI-MB07. Note the extended lag phase and partial growth promotion at low concentrations for some antibiotics. This hormetic effect was associated with higher maximum specific growth rates and increased OD values. Each data point (time resolution over 48 h: 15 min) represents mean values of triplicate cultivations, normalized with data from identical incubations in the absence of bacterial cells (sterile controls). Hence, each curve represents 576 single data points and kinetics from about 21,900 single data points are shown in this figure. For visual clarity, standard deviations (error bars) are only presented for selected time points.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Effect of selected antibiotics on the growth dynamics of Enterococcus faecium strain UKI-MB07. Note the extended lag phase and partial growth promotion at low concentrations for some antibiotics. This hormetic effect was associated with higher maximum specific growth rates and increased OD values. Each data point (time resolution over 48 h: 15 min) represents mean values of triplicate cultivations, normalized with data from identical incubations in the absence of bacterial cells (sterile controls). Hence, each curve represents 576 single data points and kinetics from about 21,900 single data points are shown in this figure. For visual clarity, standard deviations (error bars) are only presented for selected time points.
Mentions: The effects of various antibiotics on the growth dynamics of the Enterococcus clinical isolate were monitored using an automated 96-well microtiter plate assay that allowed simultaneous cultivation and on-line analysis of bacterial growth. By examining growth over time using the automated method, different effective exposure times and concentration-dependent effects on the growth dynamics of the Enterococcus clinical isolate became evident. Despite the fact that categorization of data after 48 h of incubation remains “theoretical” since we did not follow the guideline incubation time for utilizing MIC interpretative standards for Enterococcus spp. (dilution methods: 16 to 20 h, 24 h for vancomycin; Clinical and Laboratory Standards Institute [CLSI], 2012, 2013), for antibiotics tested at two concentrations, the Enterococcus isolate was sensitive to cefotaxim (2/8), doxycyclin (1/4), cotrimoxazol (16/64), mupirocin (4/8), and erythromycin (1/4) in both the 20- and 48 h experiments. According to the current breakpoint interpretation (Clinical and Laboratory Standards Institute [CLSI], 2013), these data indicate that the Enterococcus test isolate can be categorized as susceptible (S) to doxycyclin and as either (S) or of intermediate susceptibility (I) to erythromycin (“theoretical” categorization of 48-h data sets). Ciprofloxacin showed intermediate performance (growth at 1 μg mL-1 and no growth at 2 μg mL-1) in both the 20- and 48-h experiments; however, growth in the presence of 1 μg mL-1 of ciprofloxacin did not begin until about 15 h of incubation (Figure 1A).

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