Hanstein Sophia, Grochow Thomas, Mötzing Marina, Fietz Simone A, Hoffmann Ralf, Baums Christoph G, Kähl Sophie
Institute of Bacteriology and Mycology, Center for Infectious Diseases, Faculty of Veterinary Medicine, Leipzig University, Leipzig, Germany.
Institute of Veterinary Anatomy, Histology and Embryology, Faculty of Veterinary Medicine, Leipzig University, Leipzig, Germany.
Front Microbiol. 2025 Apr 11;16:1548362. doi: 10.3389/fmicb.2025.1548362. eCollection 2025.
is a major human nosocomial infectious agent and an important veterinary pathogen, frequently resistant to various antibiotics. It causes diseases such as pneumonia, urinary tract infections, surgical wound infections and septicemia. Biofilm formation of promotes persistent infection and contributes to resistance against antimicrobial agents. The objective of this study was to comparatively evaluate the effect of selected AMPs on the formation, metabolic activity and viability of biofilms of veterinary and human origin.
Biofilm formation of three strains was quantified using the crystal violet assay and visualized by scanning electron microscopy (SEM). The inhibitory effects of eight different AMPs on the formation and metabolic activity of biofilms, as well as on planktonic growth, were examined using crystal violet, resazurin and broth microdilution assays, respectively. The effect on living and dead bacteria in mature biofilms was investigated using the fluorescent dyes SYTO™ 9 and propidium iodide. In addition, the distribution of rhodamine B-labeled peptide DJK-5 in mature biofilms of strain 17349 was visualized by confocal laser scanning microscopy (CLSM).
Biofilm formation was confirmed for all three strains. Depending on the strain, we found that planktonic growth was affected by the AMPs DJK-5, DJK-6, Onc72, and Onc112. Biofilm formation of all three strains was inhibited by hbD3, LL-37, DJK-5, and DJK-6, with biofilm mass reduced to less than 40% of the untreated control. In addition to the inhibition of biofilm formation, a reduction in the metabolic activity of the biofilm-associated bacteria was also observed. These four AMPs also showed an effect on mature biofilms by reducing the number of both viable and dead bacteria in 22 h-old biofilms. Rhodamine B-labeled DJK-5 took 7 h to visibly accumulate in the planktonic bacteria. Multi-layered biofilm aggregations were mainly negative for rhodamine B-labeled DJK-5, even 44 h after AMP treatment, indicating that certain parts of mature biofilms are not accessible for this AMP.
In conclusion, we found differences in the effect of AMPs on biofilms including both increases and decreases in biofilm mass and viability.
是一种主要的人类医院感染病原体和重要的兽医病原体，经常对各种抗生素耐药。它会引发肺炎、尿路感染、手术伤口感染和败血症等疾病。生物膜的形成促进了持续性感染，并导致对抗菌剂产生耐药性。本研究的目的是比较评估选定的抗菌肽对兽医和人类来源生物膜的形成、代谢活性和活力的影响。
使用结晶紫测定法定量三株菌株的生物膜形成，并通过扫描电子显微镜（SEM）进行可视化。分别使用结晶紫、刃天青和肉汤微量稀释测定法，检测八种不同抗菌肽对生物膜形成、代谢活性以及浮游生长的抑制作用。使用荧光染料SYTO™ 9和碘化丙啶研究对成熟生物膜中活菌和死菌的影响。此外，通过共聚焦激光扫描显微镜（CLSM）观察罗丹明B标记的肽DJK-5在17349菌株成熟生物膜中的分布。
确认所有三株菌株均能形成生物膜。根据菌株不同，我们发现浮游生长受到抗菌肽DJK-5、DJK-6、Onc72和Onc112的影响。所有三株菌株的生物膜形成均受到hbD3、LL-37、DJK-5和DJK-6的抑制，生物膜量减少至未处理对照的40%以下。除了抑制生物膜形成外，还观察到生物膜相关细菌的代谢活性降低。这四种抗菌肽对成熟生物膜也有作用，可减少22小时龄生物膜中活菌和死菌的数量。罗丹明B标记的DJK-5需要7小时才能在浮游细菌中明显积累。多层生物膜聚集体对罗丹明B标记的DJK-5主要呈阴性，即使在抗菌肽处理44小时后也是如此，这表明成熟生物膜的某些部分无法被这种抗菌肽触及。
总之，我们发现抗菌肽对生物膜的影响存在差异，包括生物膜量和活力的增加和减少。
