Muehler Denise, Rupp Christina M, Keceli Sercan, Brochhausen Christoph, Siegmund Heiko, Maisch Tim, Hiller Karl-Anton, Buchalla Wolfgang, Cieplik Fabian
Department of Conservative Dentistry and Periodontology, University Hospital Regensburg, Regensburg, Germany.
Institute of Pathology, University Hospital Regensburg, Regensburg, Germany.
Front Microbiol. 2020 Oct 30;11:589364. doi: 10.3389/fmicb.2020.589364. eCollection 2020.
In view of increasing resistance against antibiotics and antiseptics, antimicrobial photodynamic therapy (aPDT) may be a promising approach for use in dentistry. The aim of this study was to investigate the mechanism of action of aPDT with the phenalene-1-one derivatives SAPYR and SA-PN-05 as photosensitizers by evaluating bacterial ability to replicate, membrane integrity, metabolic activity, and formation of reactive oxygen species (ROS) in biofilms of , , and .
Single-species biofilms (, , and ) were cultured under aerobic conditions for 48 h followed by treatment with the photosensitizers SAPYR and SA-PN-05 at various concentrations (0, 50, 100, 500 μM) and different incubation periods of 5, 10, 20, and 30 min and subsequent irradiation for 10 min (Waldmann PIB 3000; λ = 360-600 nm; 50 mW/cm; 30 J/cm). Control samples were treated with dHO and kept in dark for the same periods. Bacterial ability to replicate was evaluated by colony forming unit (CFU) assay. The cytoplasmic membrane integrity was investigated by flow cytometry using SYBR Green and propidium iodide and visualized by scanning and transmission electron microscopy. For SAPYR, metabolic activity and formation of intracellular ROS after irradiation were evaluated via luminescence and fluorometric assays, respectively.
SAPYR showed antimicrobial effects (>3 log CFU reduction) on after 5 min and on after 20 min incubation and light activation. For , CFU reduction was >2 log after 30 min of incubation. SA-PN-05 showed an antimicrobial effect after 5 min for all bacteria. Membrane damage upon aPDT with SAPYR was observed for , but not for and . Following treatment with SA-PN-05, irradiated samples and dark controls of all three species showed loss of membrane integrity. Luminescence and fluorometric assays showed a reduction in metabolic activity and an increase in formation of intracellular ROS in all three species upon aPDT treatment with SAPYR.
The observed loss in ability to replicate upon aPDT with SAPYR in single-species biofilms may be due to an increase in formation of intracellular ROS upon photodynamic treatment.
鉴于对抗生素和防腐剂的耐药性不断增加，抗菌光动力疗法（aPDT）可能是牙科领域一种有前景的方法。本研究的目的是通过评估细菌的复制能力、膜完整性、代谢活性以及在变形链球菌、嗜酸乳杆菌和血链球菌生物膜中活性氧（ROS）的形成，来研究以菲-1-酮衍生物SAPYR和SA-PN-05作为光敏剂的aPDT的作用机制。
单菌种生物膜（变形链球菌、嗜酸乳杆菌和血链球菌）在有氧条件下培养48小时，然后用不同浓度（0、50、100、500μM）的光敏剂SAPYR和SA-PN-05处理，并在5、10、20和30分钟的不同孵育时间后进行10分钟的照射（Waldmann PIB 3000；λ = 360 - 600nm；50mW/cm²；30J/cm²）。对照样品用去离子水（dHO）处理，并在相同时间内置于黑暗中。通过菌落形成单位（CFU）测定评估细菌的复制能力。使用SYBR Green和碘化丙啶通过流式细胞术研究细胞质膜完整性，并通过扫描和透射电子显微镜观察其可视化情况。对于SAPYR，分别通过发光和荧光测定评估照射后细胞的代谢活性和细胞内ROS的形成。
SAPYR在孵育5分钟后对变形链球菌、孵育20分钟并光照激活后对嗜酸乳杆菌显示出抗菌效果（CFU减少>3 log）。对于血链球菌，孵育30分钟后CFU减少>2 log。SA-PN-05在孵育5分钟后对所有细菌均显示出抗菌效果。在用SAPYR进行aPDT后，观察到变形链球菌的膜损伤，但嗜酸乳杆菌和血链球菌未观察到。在用SA-PN-05处理后，所有三种菌种的照射样品和黑暗对照均显示膜完整性丧失。发光和荧光测定显示，在用SAPYR进行aPDT处理后，所有三种菌种的代谢活性降低，细胞内ROS形成增加。
在用SAPYR对单菌种生物膜进行aPDT后观察到的复制能力丧失可能是由于光动力处理后细胞内ROS形成增加所致。
