智能高通量抗菌药敏检测/噬菌体筛选系统及Lar耐药性指数的应用
1Shandong Key Laboratory of Animal Disease Control and Breeding, Institute of Animal Science and Veterinary Medicine, Shandong Academy of Agricultural Sciences, P.R. China,Key Laboratory of Livestock and Poultry Multi-omics of MARA, 2Department of Genetic and Genomes Biology,University of Leicester, 3College of Veterinary Medicine,Nanjing Agricultural University
Summary
在这里，我们介绍了智能高通量抗菌药敏检测/噬菌体筛选系统的原理、结构和说明。以中国山东省家禽中分离的 沙门氏菌 为例，说明了其应用。计算Lar指数，并全面讨论其在评估抗菌素耐药性方面的意义。
Abstract
为了提高耐药菌药敏试验（AST）和噬菌体高通量筛选的效率，降低检测成本，根据AST标准和临床与实验室标准协会（CLSI）制定的耐药性断点（R），开发了一种智能高通量AST/噬菌体筛选系统，包括96点阵接种器、图像采集转换器和相应的软件。采用智能高通量AST/噬菌体筛选系统，对山东省家禽中分离的1500株 沙门氏 菌菌株的AST和最小抑菌浓度（MIC）分布（R/8-8R）进行统计。Lar指数，即“较少的抗生素，较少的耐药性和残留，直到很少的抗生素”，是通过计算每个MIC的加权平均值并除以R得到的。与使用耐药性流行率来表征高度耐药菌株的抗菌素耐药性 （AMR） 程度相比，这种方法提高了准确性。对于AMR高的 沙门氏 菌菌株，利用该系统从噬菌体文库中高效筛选裂解噬菌体，并计算分析裂解谱图。结果表明，智能高通量AST/噬菌体筛选系统具有可操作性、准确性、高效、成本低廉、易于维护等特点。该系统与山东省兽用抗菌素耐药性监测系统相结合，适用于与AMR相关的科学研究和临床检测。
Introduction
随着抗菌药物被广泛用于预防细菌性传染病，抗菌素耐药性（AMR）已成为全球公共卫生问题1。抗击抗微生物药物耐药性是目前监测流行病病原体抗微生物药物耐药性以及敏感抗菌剂和溶菌噬菌体的协同治疗的主要任务2。
体外 抗菌药物敏感性试验 （AST） 是监测治疗和检测 AMR 水平的主要方法。它是抗菌药理学的重要组成部分，也是临床用药的重要基础。美国临床和实验室标准协会（CLSI）和欧洲药敏试验委员会（EUCAST）制定和修订了AST的国际标准，并不断修改和补充AST方法和断点，以确定某种“生物体-抗菌剂”组合的MIC为敏感（S）、耐药（R）或中间（I）3，4.
从 1980 年代到 1990 年代，自动微量肉汤稀释仪器迅速发展并应用于临床实践，例如 Alfred 60AST、VITEK System、FISTM 和 Cobasbact5、6、7。然而，这些仪器价格昂贵，需要高成本的耗材，并且它们的检测范围是为临床患者用药设计的5,6,7。由于这些原因，它们不适合兽医临床检查和大量高耐药菌株的检测。本研究开发了一种智能高通量AST/噬菌体筛选系统，包括96点基质接种器（图1）、图像采集转换器（图2）和相应的软件8，通过琼脂稀释法对一批细菌菌株同时对多种抗菌剂进行AST。此外，该系统还用于检测和分析噬菌体对抗菌耐药菌的裂解模式9，并从噬菌体库中高效筛选裂解噬菌体。该系统被发现高效、经济且易于操作。
图 1：96 点阵接种器的结构图。 1：接种针板;2：移动运营商;3：种子块;4：孵育板;5：底座;6：操作手柄;7：限位销。请点击这里查看此图的较大版本.
图2：图像采集转换器的结构图。 1：外壳;2：显示屏;3：图像采集室;4：检测板底座;5：出入库检测板;6：控制板;7：图像采集转换装置;8：光源;9：图像扫描仪。请点击这里查看此图的较大版本.
Protocol
本研究中使用的 沙门氏菌菌 株是在获得中国山东省农业科学院畜牧兽医研究所生物安全委员会的批准后从中国山东家禽中采集的。 1. 智能高通量AST系统的应用8 接种物制备将质量控制微生物 大肠杆 菌和93种 沙门氏菌 菌株在37°C下在Mueller-Hinton琼脂（MHA）平板上检测AST16-18小时3。 根据C…
Representative Results
遵循智能高通量AST系统的方案，以中国山东家禽 沙门氏菌 为例说明其应用。 沙 门氏菌 菌株在含有氨苄青霉素（R为32μg/ mL）的琼脂平板上生长，浓度为2至256μg/ mL，由图像采集转换器确定，如图 3所示。水平1号 井A1为阴性对照，未显示菌落生长;A2和A3为CLSI标准化的质控范围（2-8 μg/mL）为质控菌株，MIC为4 μg/mL（用2 μg/mL氨苄青霉素…
Discussion
琼脂稀释法已经得到广泛应用。高通量AST系统的原理是琼脂稀释法。该协议中的关键步骤之一是一次准确高通量转移 96 个接种物，该转移连续多次进行。为了完成这一关键步骤，96点阵接种器的引脚均匀且非常光滑。每个引脚的自然沉积体积约为 2 μL，在琼脂培养基表面聚集成小液滴，迅速吸收到琼脂中，不会流动或飞溅以造成交叉污染。第二个关键步骤是对大型AST数据进行统计处理。在高通量?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
本工作得到国家重点研发专项（2019YFA0904003）资助;山东省现代农业产业体系（SDAIT-011-09）;国际合作平台优化项目（CXGC2023G15）;山东省农业科学院农业科技创新项目重大创新任务（CXGC2023G03）.
Materials
| 96 well culture plate | Beijing lanjieke Technology Co., Ltd | 11510 | |
| 96-dot matrix AST image acquisition system | Institute of Animal Science and Veterinary Medicine, Shandong Academy of Agricultural Sciences | In-house software copyright | |
| 96-dot matrix inoculator | Institute of Animal Science and Veterinary Medicine, Shandong Academy of Agricultural Sciences | N/A | Patented product |
| Agar | Qingdao hi tech Industrial Park Haibo Biotechnology Co., Ltd | HB8274-1 | |
| Amikacin | Shanghai McLean Biochemical Technology Co., Ltd | A857053 | |
| Amoxicillin | Shanghai McLean Biochemical Technology Co., Ltd | A822839 | |
| Ampicillin | Shanghai McLean Biochemical Technology Co., Ltd | A830931 | |
| Analytical balance | Sartorius | BSA224S | |
| Automated calculation software for Lar index of AMR | Institute of Animal Science and Veterinary Medicine, Shandong Academy of Agricultural Sciences | In-house software copyright | |
| Bacteria Salmonella strains | Institute of Animal Science and Veterinary Medicine, Shandong Academy of Agricultural Sciences | N/A | Animal origin |
| Bacterial resistance Lar index certification management system V1.0 | Institute of Animal Science and Veterinary Medicine, Shandong Academy of Agricultural Sciences | In-house software copyright | |
| Ceftiofur | Shanghai McLean Biochemical Technology Co., Ltd | C873619 | |
| Ciprofloxacin | Shanghai McLean Biochemical Technology Co., Ltd | C824343 | |
| Clavulanic acid | Shanghai McLean Biochemical Technology Co., Ltd | C824181 | |
| Clean worktable | Suzhou purification equipment Co., Ltd | SW-CJ-2D | |
| Colistin sulfate | Shanghai McLean Biochemical Technology Co., Ltd | C805491 | |
| Culture plate | Institute of Animal Science and Veterinary Medicine, Shandong Academy of Agricultural Sciences | N/A | Patented product |
| Doxycycline | Shanghai McLean Biochemical Technology Co., Ltd | D832390 | |
| Enrofloxacin | Shanghai McLean Biochemical Technology Co., Ltd | E809130 | |
| Filter 0.22 μm | Millipore | SLGP033RB | |
| Florfenicol | Shanghai McLean Biochemical Technology Co., Ltd | F809685 | |
| Gentamicin | Shanghai McLean Biochemical Technology Co., Ltd | G810322 | |
| Glass bottle 50 mL | Xuzhou Qianxing Glass Technology Co., Ltd | QX-7 | |
| High-throughput resistance detection system V1.0 | Institute of Animal Science and Veterinary Medicine, Shandong Academy of Agricultural Sciences | In-house software copyright | |
| Image acquisition converter | Institute of Animal Science and Veterinary Medicine, Shandong Academy of Agricultural Sciences | N/A | Patented product |
| Meropenem | Shanghai McLean Biochemical Technology Co., Ltd | M861173 | |
| Mueller-Hinton agar | Qingdao hi tech Industrial Park Haibo Biotechnology Co., Ltd | HB6232 | |
| Petri dish 60 mm x 15 mm | Qingdao Jindian biochemical equipment Co., Ltd | 16021-1 | |
| Petri dish 90 mm x 15 mm | Qingdao Jindian biochemical equipment Co., Ltd | 16001-1 | |
| Salmonella phages | Institute of Animal Science and Veterinary Medicine, Shandong Academy of Agricultural Sciences | N/A | |
| Shaker incubator | Shanghai Minquan Instrument Co., Ltd | MQD-S2R | |
| Turbidimeter | Shanghai XingBai Biotechnology Co., Ltd | F-TC2015 | |
| Varms base type library system V1.0 | Institute of Animal Science and Veterinary Medicine, Shandong Academy of Agricultural Sciences | In-house software copyright | |
| Vertical high-pressure steam sterilizer | Shanghai Shen'an medical instrument factory | LDZX-75L | |
| Veterinary pathogen resistance testing management system | Institute of Animal Science and Veterinary Medicine, Shandong Academy of Agricultural Sciences | In-house software copyright | |
| Veterinary resistance cloud monitoring and phage control platform V1.0 | Institute of Animal Science and Veterinary Medicine, Shandong Academy of Agricultural Sciences | In-house software copyright |
Riferimenti
- Ramanan, L., et al. Antimicrobial resistance-the need for global solutions. The Lancet Infectious Diseases. 13 (12), 1057-1098 (2013).
- Xiaonan, Z., Qing, Z., Thomas, S. P., Yuqing, L., Martha, R. J. C. inPhocus: Perspectives of the application of bacteriophages in poultry and aquaculture industries based on Varms in China. PHAGE: Therapy, Applications, and Research. 2 (2), 69-74 (2021).
- CLSI. . Performance Standards for Antimicrobial Disk Susceptibility Tests. CLSI document M100. , (2022).
- Yuqing, L., et al. . Antimicrobial Sensitivity Testing Standard of EUCAST. , (2017).
- Barnini, S., et al. A new rapid method for direct antimicrobial susceptibility testing of bacteria from positive blood cultures. BMC Microbiology. 16 (1), 185-192 (2016).
- Höring, S., Massarani, A. S., Löffler, B., Rödel, J. Rapid antimicrobial susceptibility testing in blood culture diagnostics performed by direct inoculation using the VITEK®-2 and BD PhoenixTM platforms. European Journal of Clinical Microbiology & Infectious Diseases. 38 (3), 471-478 (2019).
- Dupuis, G. Evaluation of the Cobasbact automated antimicrobial susceptibility testing system. European Journal of Clinical Microbiology & Infectious Diseases. 4 (2), 119-122 (1985).
- Liu, Y., et al. A system of bacterial antimicrobial resistance detection and its operation method. China Patent. , (2019).
- Liu, Y. A high throughput test plate for screening bacteriophage of zoonotic pathogens and its application. China Patent. , (2022).
- Adams, M. H. . Bacteriophages. , (1959).
- Nair, A., Ghugare, G. S., Khairnar, K. An appraisal of bacteriophage isolation techniques from environment. Microbial Ecology. 83 (3), 519-535 (2022).
- . . Shandong veterinary antibiotic resistance system. , (2023).
- Ming, H., et al. Comparison of the results of 96-dot agar dilution method and broth microdilution method. Chinese Journal of Antibiotics. 43 (6), 729-733 (2018).
- Laxminarayan, R., Klugman, K. P. Communicating trends in resistance using a drug resistance index. BMJ Open. 1 (2), e000135 (2011).
- Chen, Y., et al. Assessing antibiotic therapy effectiveness against the major bacterial pathogens in a hospital using an integrated index. Future Microbiology. 12, 853-866 (2017).
- Ciccolini, M., Spoorenberg, V., Geerlings, S. E., Prins, J. M., Grundmann, H. Using an index-based approach to assess the population-level appropriateness of empirical antibiotic therapy. Journal of Antimicrobial Chemotherapy. 70 (1), 286-293 (2015).
- Yanbo, L., et al. Preliminary application of inoculation system for high-throughput drug susceptibility test. China Poultry. 42 (6), 52-57 (2020).
Citazione di questo articolo
Hu, M., Liu, Z., Song, Z., Li, L., Zhao, X., Luo, Y., Zhang, Q., Chen, Y., Xu, X., Dong, Y., Hrabchenko, N., Zhang, W., Liu, Y. Application of the Intelligent High-Throughput Antimicrobial Sensitivity Testing/Phage Screening System and Lar Index of Antimicrobial Resistance. J. Vis. Exp. (197), e64785, doi:10.3791/64785 (2023).