Department of Clinical Microbiology, Rigshospitalet, 2100 Copenhagen, Denmark; Department of Immunology and Microbiology, Costerton Biofilm Center UC-CARE, Faculty of Health Sciences, University of Copenhagen, 2200 Copenhagen, Denmark.
Biomedical Engineering, Department of Electrical Engineering, Technical University of Denmark, 2800 Lyngby, Denmark; Biomedical Engineering, Department of Health Technology, Technical University of Denmark, 2800 Lyngby, Denmark.
Int J Antimicrob Agents. 2019 May;53(5):564-573. doi: 10.1016/j.ijantimicag.2018.12.015. Epub 2019 Jan 5.
Pseudomonas aeruginosa PAO1 (tobramycin MIC = 0.064 µg/mL) was used to perform agar diffusion tests employing tobramycin-containing tablets. Bacterial growth and formation of inhibition zones were studied by stereomicroscopy and by blotting with microscope slides and staining with methylene blue, Alcian blue and a fluorescent lectin for the P. aeruginosa PSL, which was studied by confocal laser scanning microscopy. Diffusion of tobramycin from the deposit was modelled using a 3D geometric version of Fick's second law of diffusion. The time-dependent gradual increase in the minimum biofilm eradication concentration (MBEC) was studied using a Calgary Biofilm Device. The early inhibition zone was visible after 5 h of incubation. The corresponding calculated tobramycin concentration at the border was 1.9 µg/mL, which increased to 3.2 µg/mL and 6.3 µg/mL after 7 h and 24 h, respectively. The inhibition zone increased to the stable final zone after 7 h of incubation. Bacterial growth and small aggregate formation (young biofilms) took place inside the inhibition zone until the small aggregates contained less than ca. 64 cells and production of polysaccharide matrix including PSL had begun; thereafter, the small bacterial aggregates were killed by tobramycin. Bacteria at the border of the stable inhibition zone and beyond continued to grow to a mature biofilm and produced large amount of polysaccharide-containing matrix. Formation of the inhibition zone during agar diffusion antimicrobial susceptibility testing is due to a switch from a planktonic to biofilm mode of growth and gives clinically important information about the increased antimicrobial tolerance of biofilms.
铜绿假单胞菌 PAO1（妥布霉素 MIC=0.064 µg/mL）被用于进行琼脂扩散试验，使用含妥布霉素的片剂。通过立体显微镜和用显微镜载玻片擦拭以及用亚甲蓝、藻蓝蛋白和荧光假单胞菌 PSL 的凝集素染色研究细菌生长和抑制区的形成，并用共聚焦激光扫描显微镜研究 PSL。使用 Fick 第二扩散定律的 3D 几何版本模拟妥布霉素从沉积物中的扩散。使用 Calgary 生物膜装置研究最小生物膜清除浓度（MBEC）随时间的逐渐增加。孵育 5 小时后可见早期抑制区。相应的边界处妥布霉素的计算浓度为 1.9 µg/mL，在 7 小时和 24 小时时分别增加到 3.2 µg/mL 和 6.3 µg/mL。孵育 7 小时后，抑制区增加到稳定的最终区。细菌生长和小聚集体形成（年轻生物膜）发生在抑制区内，直到小聚集体中包含的细胞少于约 64 个，并且开始产生包括 PSL 的多糖基质；此后，小细菌聚集体被妥布霉素杀死。稳定抑制区边界和之外的细菌继续生长到成熟生物膜，并产生大量含有多糖的基质。琼脂扩散药敏试验中抑制区的形成是由于从浮游生物到生物膜生长模式的转变，这为生物膜增加的抗菌药物耐受性提供了重要的临床信息。
