atp 利用商业检测和染色法定量检测淋病奈瑟菌骨料的抗生素敏感性
Summary
采用简单的 atp 测量法和活死染色法对头孢曲松治疗后淋病奈瑟菌的生存率进行了定量和可视化。该方案可以扩展到检查任何抗生素的抗菌作用, 并可用于确定细菌生物膜中抗生素的最小抑制浓度。
Abstract
抗生素耐药淋病奈瑟菌 (gc) 的出现是一个世界性的健康威胁, 并突出表明需要确定未能通过治疗的个人。这革兰氏阴性细菌在人完全导致淋病。在感染过程中, 它能够形成聚集体和/或生物膜。最低抑制浓度 (mic) 测试用于确定对抗生素的敏感性, 并确定适当的治疗方法。然而, 体内根除的机制及其与实验室结果的关系尚不清楚。一种研究气相色谱聚集对抗生素敏感性的影响, 并显示聚集大小与抗生素敏感性之间的关系的方法。当 gc 集料时, 它们对抗生素的杀灭有更强的抵抗力, 中心中的细菌比外围的头孢曲松治疗效果更好。结果表明, 淋病 n 聚集可以降低其对头孢曲松的易感性, 这在标准的琼脂片 mic 方法中没有得到反映。本研究中使用的方法将使研究人员能够在临床相关条件下测试细菌敏感性。
Introduction
淋病是一种常见的性传播感染 (sti)1。淋病奈瑟菌(gc), 革兰氏阴性的双球菌细菌, 是这种疾病的病原体。生殖器感染的症状可导致排尿疼痛、全身生殖器疼痛和尿道分泌物。感染往往是无症状的 2,3,4,5, 这允许延长定植。这些未经治疗的感染是一个主要的健康问题, 因为它们有可能促进机体的传播, 这可能导致并发症, 如盆腔炎 (pid) 和传播的淋球菌感染 (dgi)6。抗生素耐药淋病是一个重大的公共卫生危机和日益沉重的社会经济负担7。头孢菌素易感性降低, 导致治疗方案从单一抗生素转变为双重治疗, 将阿奇霉素或多西环素与头孢曲松8结合起来。头孢曲松和阿奇霉素9,10的失败增加, 再加上无症状感染, 突出表明需要了解淋病治疗失败。
最低抑制浓度 (mic) 试验, 包括琼脂稀释和圆盘扩散试验, 已被用作识别抗生素耐药性的标准医学试验。然而, 目前尚不清楚 mic 测试是否反映了细菌在体内的抗生素耐药性。细菌生物膜的形成有助于细菌在抗生素的杀菌浓度存在下的生存: mic 测试无法检测到这种效果11。因为 gc 可以在黏膜表面形成生物膜 12, 我们假设聚集体中的抗生素敏感性将不同于在单个 gc 中看到的。此外, 研究表明, 三相可变表面分子, 皮利, 与蛋白相关的蛋白质 (opa) 和胶质类高糖, 调节细菌间相互作用, 导致不同大小的聚集体 13,14,15. 由于缺乏适当的方法, 这些成分对抗生素抗药性的贡献没有得到审查。
目前, 有几种方法来衡量生物膜的根除。最广泛使用的定量方法是用结晶紫罗兰色染色16测量生物量的变化。然而, 该方法需要大量的实验操作, 这可能会产生错误的实验重复17。这里使用的活体/死染色方法允许显示活细菌和死细菌及其在生物膜中的分布。然而, 生物膜结构可以作为一个物理屏障, 减少染料渗透。因此, 为了量化一个群体中的活死细菌, 染色仅限于小生物膜或其前世菌-微菌落或聚集物。其他方法, 包括琼脂稀释和圆盘扩散试验, 无法测量聚集的影响。要检测抗生素暴露后聚集中的 gc 敏感性, 一种理想的方法需要进行定量检测, 以测量活菌并显示其分布。
这里描述的程序结合了 atp 利用测量和活/死染色检测, 以定量和直观地检查在抗生素存在的集合体中的 gc 敏感性。
Protocol
Representative Results
Discussion
细菌在人体感染过程中可以形成生物膜。传统的 mic 检测可能无法反映消除生物膜中细菌所需的浓度。为了测试抗菌素对生物膜的影响, 由于生物膜结构的影响, 基于生物膜生物量和电镀 cfu 的方法可能是错误的。例如, 只有当生物膜可能被破坏时, 电镀方法才有效。因此, 获得的 cfu 可能低于实际的活细菌数量。在生物膜内发现了死亡和活细菌的视觉, 以测量生物膜内细菌的存活, 然而, 根据生物膜的…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了国家卫生研究所向 d. c. s. 和 w. s. ai123340 提供的赠款的支持。l.-c. w.、j. w.、a. c. 和 e. n. 在参与马里兰州立大学资助的 “第一年创新 & 研究经验” 项目中得到了支持。资助者在研究设计、数据收集和分析、决定出版或准备手稿方面没有任何作用。我们承认 umd cbmg 成像核心用于所有显微镜实验。
Materials
| 100x Kellogg's supplement | |||
| Agar | United States Biological | A0930 | |
| BacTiter Assay | Promega | G8232 | |
| Ceftriaxone | TCI | C2226 | |
| Difco GC medium base | BD | 228950 | |
| Ferric nitrate, nonahydrate | Sigma-Aldrich | 254223-10G | |
| Glucose | Thermo Fisher Scientific | BP350-1 | |
| L-glutamine Crystalline Powder | Fisher Scientific | BP379-100 | |
| BacLight live/dead staining | Invitrogen | L7012 | |
| MS11 Neisseria gonorrhoeae strain | kindly provided by Dr. Herman Schneider, Walter Reed Army Institute for Research | ||
| Potassium phosphate dibasic (K2HPO4) | Fisher Scientific | P290-500 | |
| Potassium phosphate monobasic (KH2PO4) | Fisher Scientific | BP329-1 | |
| Proteose Peptone | BD Biosciences | 211693 | |
| Sodium chloride (NaCl) | Fisher Scientific | S671-10 | |
| Soluble Starch | Sigma-Aldrich | S9765 | |
| Thiamine pyrophosphate | Sigma-Aldrich | C8754-5G | |
| Equipment | |||
| Petri Dishes | VWR | 25384-302 | |
| 8-well coverslip-bottom chamber | Thermo Fisher Scientific | 155411 | |
| 96-well tissue culture plates | Corning, Falcon | 3370 | |
| Biosafety Cabinet (NU-425-600 Class II, A2 Laminar Flow Biohazard Hood) | Nuaire | 32776 | |
| CO2 Incubator | Fisher Scientific | Model 3530 | |
| Confocal microscope equipped with live imaging chamber | Leica | SP5X | |
| Corning 96 Well Black Polystyrene Microplate | Corning | 3904 | |
| Glomax Illuminator | Promega | E6521 | |
| Pipette tips (0.1-10 µL) | Thermo Fisher Scientific | 02-717-133 | |
| Pipette tips (1000 µL) | VWR | 83007-382 | |
| Pipette tips (200 µL) | VWR | 53509-007 | |
| Spectrophotometer Ultrospec 2000 UV | Pharmacia Biotech | 80-2106-00 | |
| Sterile 15 ml conical tubes | VWR | 21008-216 | |
| Sterile Microcentrifuge Tubes (1.7 mL) | Sorenson BioScience | 16070 | |
| Sterile polyester-tipped applicators | Fisher Scientific | 23-400-122 | |
| Sonicator | Kontes | Equivelent to 9110001 |
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