用于结核病疫苗功效评估的微集落形成单位测定
Summary
菌落形成单位 （CFU） 的测定是定量细菌的金标准技术，包括 结核分枝杆菌，结核分枝杆 菌可能需要数周时间才能形成可见菌落。在这里，我们描述了一种用于 CFU 测定的微型 CFU，它提高了时间效率，减少了实验室空间和试剂成本，并可扩展到中高通量实验。
Abstract
结核病 （TB） 是全球传染性病原体导致死亡的主要原因，2022 年造成 160 万人死亡，仅在 2019-2021 年大流行期间被 COVID-19 超越。该病由 结核分枝杆菌 （M.tb）引起。 牛分枝杆菌 卡介苗（BCG）是唯一的结核病疫苗，是世界上获得许可的最古老的疫苗，仍在使用。目前，有12种疫苗处于临床试验阶段，数十种疫苗处于临床前开发阶段。在临床前研究中，用于评估结核病疫苗疗效的首选方法是通过菌落形成单位 （CFU） 测定法对细菌菌落进行计数。这种耗时的检测需要 4 到 6 周才能完成，需要大量的实验室和培养箱空间，试剂成本高，并且容易受到污染。在这里，我们描述了一种优化的菌落计数方法，即微 CFU （mCFU），它为分析 结核分枝杆 菌疫苗的功效结果提供了一种简单而快速的解决方案。mCFU 检测所需的试剂数量减少了 10 倍，潜伏期缩短了 3 倍，需要 1 到 2 周才能完成，减少了实验室空间和试剂成本，并将与大量 M.tb 相关的健康和安全风险降至最低。此外，为了评估结核病疫苗的功效，可以从各种来源获得样本，包括感染分枝杆菌的接种疫苗动物的组织。我们还描述了一种优化的方法，用于感染研究生产单细胞、均匀和高质量的分枝杆菌培养物。最后，我们建议将这些方法普遍用于疫苗功效测定的临床前研究，最终缩短结核病疫苗开发的时间。
Introduction
结核病（TB）是全球单一传染性病原体结 核分枝杆菌 （M.tb）死亡的主要原因，其死亡人数超过任何其他病原体。2021年，结核病导致160万人死亡，在2019-2021年大流行期间被COVID-19超过1。此外，根据世界卫生组织的 2022 年全球结核病报告，COVID-19 大流行是导致新发结核病病例增加的原因。世卫组织还报告说，在此期间，被诊断患有结核病的人数大幅下降，这可能会进一步增加结核病病例数1。
卡介苗 （BCG） 是致病性牛分枝杆菌的减毒活菌株，100 多年前首次用作疫苗。这是唯一的结核病疫苗，也是世界上仍在使用的最古老的许可疫苗2,3。目前，有 12 种疫苗处于临床试验的不同阶段4，数十种疫苗正在临床前开发中 5,6。结核病疫苗的临床前评估包括安全性和免疫原性评估7，这可以在斑马鱼、小鼠、豚鼠、兔子、牛和非人灵长类动物等各种动物模型中获得 8,9,10。此外，评估疫苗诱导对结核分枝杆菌感染和/或传播的保护能力，即疫苗效力，需要在体内进行结核分枝杆菌攻击5,11。有趣的是，卡介苗疫苗接种通过训练有素的免疫机制 12,13 诱导影响其他细菌和病毒病原体存活的非特异性效应14。为了量化受感染动物的活细菌负荷，选择的方法是通过菌落形成单位 （CFU） 测定法 5,15 对细菌菌落进行计数。CFU是一个单位，用于估计在特定生长条件下形成菌落的微生物（细菌或真菌）的数量。CFU起源于可存活和可复制的微生物，每个菌落中活微生物的绝对数量难以估计。尚不确定菌落是否起源于一种或多种微生物。CFU单位反映了这种不确定性，因此在同一样品的重复中可以观察到很大的变异性。这种耗时的检测需要经过培训的专业技术人员在生物安全 3 级 （BSL3） 设施、大量实验室和培养箱空间中工作，需要 4 到 6 周才能完成，并且容易受到污染。
在这项研究中，我们描述了一种优化的菌落计数方法，即 micro-CFU （mCFU），并提供了一种简单快速的解决方案来分析结果 15,16,17,18,19,20。mCFU 检测所需的试剂数量减少了 10 倍，潜伏期缩短了 3 倍，需要 1 到 2 周才能完成，减少了实验室空间和试剂成本，并将与大量 M.tb 相关的健康和安全风险降至最低。我们建议将这种方法普遍用于疫苗功效测定的临床前研究，最终缩短结核病疫苗的开发时间。最后，这种优化的CFU计数方法不仅用于定量分枝杆菌，还用于定量其他细菌，如大肠杆菌和Ralstonia solanacearum21。
Protocol
注意：此处描述的方案适用于BCG，但可以应用于任何分枝杆菌。当 BSL3 设施不可用时，BCG 可用作结核病实验的替代细菌22.使用卡介苗的以下程序应在生物安全 2 级 （BSL2） 实验室下进行，并遵循适当的生物安全指南和操作危险组 2 微生物的良好实验室规范。 1. 培养基制备 根据供应商的说明，准备补充有 10% （v/v） 油酸、白蛋白、葡萄糖…
Representative Results
这里描述的mCFU测定将可以从单个培养皿中检索到的信息量增加到至少96倍。图 5 描述了两种药物递送方法的比较，用于重新利用沙奎那韦 （SQV）31,32 作为宿主定向药物治疗结核病。在该测定中，使用四种不同的结核分枝杆菌菌株感染原代人巨噬细胞。结核分枝杆菌葡萄牙国立卫生研究院的Ricardo Jorge博士（INSA）从活?…
Discussion
结核病是一个重要的公共卫生问题，其重要性日益增加，特别是在低收入和中等收入国家。在COVID-19大流行期间，诊断和治疗结核病的医疗机构中断，对新发病例的发病率产生了负面影响1。此外，必须紧急解决多药和广泛耐药的结核分枝杆菌菌株以及结核分枝杆菌和艾滋病毒的合并感染，以控制这一流行病1,34。替代或改?…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项工作得到了葡萄牙天主教大学医学院的内部资金和科学与技术基金会 （FCT） 的外部资金支持，赠款为 UIDP/04279/2020、UIDB/04279/2020 和 EXPL/SAU-INF/0742/2021。
Materials
| 96-well plates | VWR | 734-2781 | |
| DSLR 15-55 mm lens | Nikon | AF-P DX NIKKOR 18-55mm f/3.5-5.6G VR | |
| DSLR camera | Nikon | D3400 | |
| DSLR macro lens | Sigma | MACRO 105mm F2.8 EX DG OS HSM | |
| Fetal calf serum | Gibco | 10270106 | |
| Fiji Software | https://fiji.sc/ | Fiji is an open-source software supported by several laboratories, institutions, and individuals. All the required plugins are included. | |
| Igepal CA-630 | Sigma-Aldrich | 18896 | |
| L-glutamine | Gibco | 25030-081 | |
| Middlebrook 7H10 | BD | 262710 | |
| Middlebrook 7H9 | BD | 271310 | |
| Multichannel pipette (0.5 – 10 µl) | Gilson | FA10013 | |
| Multichannel pipette (20 – 200 µl) | Gilson | FA10011 | |
| Mycobacterium bovis BCG | American Type Culture Collection | ATCC35734 | strain TMC 1011 [BCG Pasteur] |
| OADC enrichment | BD | 211886 | |
| Phosphate buffered saline (PBS) | NZYTech | MB25201 | |
| RPMI 1640 medium | Gibco | 21875091 | |
| Sodium pyruvate | Gibco | 11360-070 | |
| Spectrophotometer UV-6300PC | VWR | 634-6041 | |
| Square Petri dish 120 x 120 mm | Corning | BP124-05 | |
| Tyloxapol | Sigma-Aldrich | T8761 | |
| Ultrasound bath Elma P 30 H | VWR | 142-0051 |
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Citar este artigo
Pires, D., Bettencourt, P. J. G. Micro-Colony Forming Unit Assay for Efficacy Evaluation of Vaccines Against Tuberculosis. J. Vis. Exp. (197), e65447, doi:10.3791/65447 (2023).