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.

Aims: Engineered metal nanoparticles are increasingly used in consumer products, in part as additives that exhibit advantageous antimicrobial properties. Conventional nanoparticle susceptibility testing is based largely on determination of nontemporal growth profiles such as measurements of inhibition zones in common agar diffusion tests, counting of colony-forming units, or endpoint or regular-interval growth determination via optical density measurements. For better evaluation of the dynamic effects from exposure to nanoparticles, a cultivation-based assay was established in a 96-well format and adapted for time-resolved testing of the effects of nanoparticles on microorganisms. Methods and Results: The modified assay allowed simultaneous cultivation and on-line analysis of microbial growth inhibition. The automated highthroughput assay combined continuous monitoring of microbial growth with the analysis of many replicates and was applied to Cupriavidus necator H16 test organisms to study the antimicrobial effects of spherical silver [Ag(0)] nanoparticles (primary particle size distribution D90 < 15 nm). Ag(0) concentrations above 80 lg ml À1 resulted in complete and irreversible inhibition of microbial growth, whereas extended lag phases and partial growth inhibition were observed at Ag(0) concentrations between 20 and 80 lg ml À1 .Addition of Ag(0) nanoparticles at different growth stages led to either complete inhibition (addition of 40 lg ml À1 Ag(0) from 0 h to 6 h) or resulted in full recovery (40 lg ml À1 Ag(0) addition ! 9 h). Conclusions: Contrary to the expected results, our data indicate growth stimulation of C. necator at certain Ag(0) nanoparticle concentrations, as well as varying susceptibility to nanoparticles at different growth stages. Significance and Impact of the Study: These results underscore the need for time-resolved analyses of microbial growth inhibition by Ag(0) nanoparticles. Due to the versatility of the technique, the assay will likely complement existing microbiological methods for cultivation and diagnostics of microbes, in addition to tests of other antimicrobial nanoparticles.

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Freely dissolved aqueous concentration and chemical activity are important determinants of contaminant transport, fate, and toxic potential. Both parameters are commonly quantified using Solid Phase Micro-Extraction (SPME) based on a sorptive polymer such as polydimethylsiloxane (PDMS). This method requires the PDMS-water partition constants, KPDMSw, or activity coefficient to be known. For superhydrophobic contaminants (log KOW >6), application of existing methods to measure these parameters is challenging, and independent measures to validate KPDMSw values would be beneficial. We developed a simple, rapid method to directly measure PDMS solubilities of solid contaminants, SPDMS(S), which together with literature thermodynamic properties was then used to estimate KPDMSw and activity coefficients in PDMS. PDMS solubility for the test compounds (log KOW 7.2-8.3) ranged over 3 orders of magnitude (4.1-5700 μM), and was dependent on compound class. For polychlorinated biphenyls (PCBs) and polychlorinated dibenzo-p-dioxins (PCDDs), solubility-derived KPDMSw increased linearly with hydrophobicity, consistent with trends previously reported for less chlorinated congeners. In contrast, subcooled liquid PDMS solubilities, SPDMS(L), were approximately constant within a compound class. SPDMS(S) and KPDMSw can therefore be predicted for a compound class with reasonable robustness based solely on the class-specific SPDMS(L) and a particular congener's entropy of fusion, melting point, and aqueous solubility.