Institute of Applied Microbiology, Justus Liebig University of Giessen, Giessen, Germany.
J Appl Microbiol. 2013 Jan;114(1):25-35. doi: 10.1111/jam.12000. Epub 2012 Nov 8.
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 micro-organisms.
The modified assay allowed simultaneous cultivation and on-line analysis of microbial growth inhibition. The automated high-throughput 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 μg ml(-1) 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 μg ml(-1) . Addition of Ag(0) nanoparticles at different growth stages led to either complete inhibition (addition of 40 μg ml(-1) Ag(0) from 0 h to 6 h) or resulted in full recovery (40 μg ml(-1) Ag(0) addition ≥9 h).
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.
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.
工程金属纳米粒子越来越多地用于消费产品，部分原因是它们作为添加剂表现出有利的抗菌特性。传统的纳米颗粒敏感性测试主要基于确定非时间性生长曲线，例如在普通琼脂扩散测试中测量抑菌区、计算集落形成单位，或通过光密度测量进行终点或定期间隔生长测定。为了更好地评估暴露于纳米颗粒的动态影响，建立了基于培养的 96 孔格式测定法，并将其适用于纳米颗粒对微生物影响的时间分辨测试。
修改后的测定法允许同时培养和在线分析微生物生长抑制。自动化高通量测定法将微生物生长的连续监测与许多重复分析相结合，并应用于铜绿假单胞菌 H16 测试生物，以研究球形银[Ag(0)]纳米颗粒（初级粒径分布 D90<15nm）的抗菌效果。Ag(0)浓度高于 80μgml(-1)导致微生物生长完全和不可逆抑制，而在 Ag(0)浓度为 20 至 80μgml(-1)之间观察到延长的迟滞期和部分生长抑制。在不同生长阶段添加 Ag(0)纳米颗粒导致完全抑制（在 0 至 6 小时添加 40μgml(-1)Ag(0)）或完全恢复（在 9 小时后添加 40μgml(-1)Ag(0)）。
与预期结果相反，我们的数据表明，在某些 Ag(0)纳米颗粒浓度下，铜绿假单胞菌的生长受到刺激，以及在不同生长阶段对纳米颗粒的敏感性不同。
这些结果强调了需要对 Ag(0)纳米颗粒对微生物生长抑制进行时间分辨分析。由于该技术的多功能性，该测定法可能会补充现有的微生物培养和诊断方法，以及其他抗菌纳米颗粒的测试。
