Königs Alexa M, Flemming Hans-Curt, Wingender Jost
Department of Aquatic Microbiology - Biofilm Centre, University Duisburg-Essen, Essen Germany.
Front Microbiol. 2015 May 5;6:395. doi: 10.3389/fmicb.2015.00395. eCollection 2015.
The antimicrobial properties of silver nanoparticles (AgNPs) have raised expectations for the protection of medical devices and consumer products against biofilms. The effect of silver on bacteria is commonly determined by culture-dependent methods. It is as yet unknown if silver-exposed bacteria can enter a metabolically active but non-culturable state. In this study, the efficacy of chemically synthesized AgNPs and silver as silver nitrate (AgNO3) against planktonic cells and biofilms of Pseudomonas aeruginosa AdS was investigated in microtiter plate assays, using cultural as well as culture-independent methods. In liquid medium, AgNPs and AgNO3 inhibited both planktonic growth and biofilm formation. The efficacy of AgNPs and AgNO3 against established, 24 h-old biofilms and planktonic stationary-phase cells was compared by exposure to silver in deionized water. Loss of culturability of planktonic cells was always higher than that of the attached biofilms. However, resuspended biofilm cells became more susceptible to AgNPs and AgNO3 than attached biofilms. Thus, the physical state of bacteria within biofilms rendered them more tolerant to silver compared with the planktonic state. Silver-exposed cells that had become unculturable still displayed signs of viability: they contained rRNA, determined by fluorescent in situ hybridization, as an indicator for potential protein synthesis, maintained their membrane integrity as monitored by differential live/dead staining, and displayed significant levels of adenosine triphosphate. It was concluded that AgNPs and AgNO3 in concentrations at which culturability was inhibited, both planktonic and biofilm cells of P. aeruginosa were still intact and metabolically active, reminiscent of the viable but non-culturable state known to be induced in pathogenic bacteria in response to stress conditions. This observation is important for a realistic assessment of the antimicrobial properties of AgNPs.
银纳米颗粒（AgNPs）的抗菌特性引发了人们对医疗设备和消费品抵御生物膜的期望。银对细菌的影响通常通过依赖培养的方法来确定。目前尚不清楚接触银的细菌是否会进入代谢活跃但不可培养的状态。在本研究中，使用培养法和非培养法，通过微孔板试验研究了化学合成的AgNPs和作为硝酸银（AgNO₃）的银对铜绿假单胞菌AdS浮游细胞和生物膜的功效。在液体培养基中，AgNPs和AgNO₃均抑制浮游生长和生物膜形成。通过在去离子水中接触银，比较了AgNPs和AgNO₃对已形成的24小时龄生物膜和浮游静止期细胞的功效。浮游细胞的可培养性丧失总是高于附着生物膜的可培养性丧失。然而，重新悬浮的生物膜细胞比附着的生物膜对AgNPs和AgNO₃更敏感。因此，与浮游状态相比，生物膜内细菌的物理状态使其对银更具耐受性。已不可培养的接触银的细胞仍显示出存活迹象：通过荧光原位杂交确定它们含有rRNA，作为潜在蛋白质合成的指标，通过差异活/死染色监测维持其膜完整性，并显示出显著水平的三磷酸腺苷。得出的结论是，在抑制可培养性的浓度下，AgNPs和AgNO₃使铜绿假单胞菌的浮游细胞和生物膜细胞仍然完整且代谢活跃，这让人联想到已知在致病细菌中因应激条件而诱导的活但不可培养状态。这一观察结果对于实际评估AgNPs的抗菌特性很重要。
