阿米卡星保护测定，用于大 肠杆菌 细胞入侵的定量与可视化
July 3rd, 2026
相继入侵的大 肠杆 菌株中的抗生素耐药性，使得利用传统庆大霉素保护检测评估细菌入侵情况面临挑战。描述了一种改进的方法，通过计数菌落形成单元定量评估细菌入侵，并通过免疫荧光共聚焦显微镜对感染细胞进行定性评估。
我们重点研究了附着性入侵性大肠杆菌如何侵入肠道上皮细胞，并识别参与这一过程的细菌因素。抗甘大霉素的分离株限制了传统的入侵检测。这种基于阿米卡辛的方案能够准确定量细胞内细菌。
首先，收集入侵检测所需的所有试剂和培养基。接着，从80摄氏度的冷冻库取出粘着侵入性鞘骨菌（AIEC）菌株的冷冻甘油库存。使用无菌木棒或移液器尖端，轻轻刮除茎上的细菌，并划线到Luria-Bertani（LB）螺旋输送板上，获得单一菌落。
将平板在37摄氏度下孵化过夜。第二天，使用无菌木棒或移液器尖端，从螺旋板上取下一块菌落。将菌落接种到3毫升LB汤中，并在37摄氏度、每分钟200转的环境下孵育一夜。
然后将过夜培养液稀释1到10毫克，浸泡在1毫升LB汤中。使用分光光度计测量稀释培养物600纳米下的光学密度。将100微升过夜培养液转移到1.5毫升微离心管中，并在6000克压力下离心机，温度为4摄氏度，持续10分钟。
小心抽取上清液，勿扰动细胞沉淀，并将沉淀重新悬浮于1毫升血清和无抗生素-抗真菌剂培养基中。计算达到感染倍数（MOI）为10所需的细菌悬浮体积。为准备感染培养基，将计算出的细菌悬浮液体积转移到无菌的1.5毫升微离心管中，并用血清和无抗生素-抗真菌药的培养基将体积补制成1毫升。
轻柔地搅拌，上下移液几次。准备96孔板，通过在无菌1X PBS中进行十倍连续稀释，确定稀释过夜细菌培养中每毫升的菌落形成单位。将200微升稀释过的过夜培养液转移到平板的第一孔。
将20微升培养液移入下一排，其中180微升1X PBS。通过移液法上下混合10次，然后用新的移液器尖端重复稀释过程，直到稀释到10的6的幂。现在将10微升点稀释液从10到2的幂次方三份，按3份压板压在LB螺旋板上。
让斑点在靠近火焰的地方被螺旋输送器吸收。将板材在27至30摄氏度的温度下孵育一夜，以防止菌落过度生长并获得清晰的单个菌落。从培养箱中取出在胶原蛋白包覆24孔培养板中培养的健康融合Caco-2上皮细胞单层，并从每个孔中抽取完整培养基。
每孔移液一毫升血清和无抗生素-抗真菌的培养基。吸入介质以去除介质痕迹，再重复洗涤一次，总共洗两次。现在，将1毫升先前制备的含AIEC感染培养基加入24孔板中每个含有Caco-2细胞的孔中。
将24孔板在室温下以500克离心15分钟，以同步细菌与上皮单层接触，并在37摄氏度与5%二氧化碳下孵育三小时，以便细菌入侵。通过在预热血清和无抗生素-无菌药培养基中加入每毫升240微克阿米卡星，制备含抗生素培养基。感染期结束后，从Caco-2细胞单层中抽取培养基，并用1毫升血清和无抗生素-抗真菌药培养基清洗每个培养基。
吸取介质后重复洗涤，总共洗两次。然后将含阿米卡辛的培养基移液1毫升，含240微克/毫升，移液器进入每个孔。同时，用100微克/毫升的庆大霉素处理另一组细胞进行对比，并在37摄氏度与5%二氧化碳的环境下培养细胞1小时，以消除细胞外细菌。
孵化后，从每个井中抽取介质。添加1毫升PBS以冲洗附着细菌。吸取PBS后再重复一次洗涤，总共洗两次。
现在将200微升稀释PBS的1%Triton X100加入每个槽，并在轨道振动器上以每分钟90转的速度培养10分钟，以裂解上皮细胞。每孔上下移液管大约30次，以将细胞从板上分离。接着，使用PBS对细胞裂解液进行10倍连续稀释。
将10微升点状稀释液从未稀释的三倍份，按3次方的比例从未稀释的3次方印在LB螺旋输送板上。斑点干燥后，在27至30摄氏度下孵育一夜，以获得清晰的单个菌落。孵育后，取出平板，从适当稀释点中计数，这些稀释点在三重组中每个点上有超过七个菌落。
最后，利用方程计算接种物和每毫升的菌落形成单位和入侵百分比。在感染侵袭性AIEC株NRG857c或非侵入性大肠杆菌DH5α对照株感染Caco-2细胞单层后，使用庆大霉或阿米卡星治疗，均出现类似的侵袭特征。这些发现表明阿米卡辛在消灭细胞外细菌方面与庆大霉素相当，并准确反映了细胞内细菌负荷。
对从炎症性肠病患者中获得的耐药大肠杆菌分离株用庆大霉素治疗，导致细菌恢复值变化极大，范围从约21%到2,788%不等。相比之下，阿米卡辛治疗显著降低了所有分离株的细菌恢复率，证明了其有效性并实现了更可靠的入侵测量。该方案能够准确定量细胞内细菌并评估上皮细胞中的细菌入侵情况。免疫荧光显微镜等其他方法可用于进一步验证细菌入侵和细胞内定位。
未来研究有望识别新的毒力因子，并探讨在随袭性大肠杆菌感染期间被靶向的宿主通路。
Overview
This article presents an alternative amikacin-based protection assay for quantifying and visualizing epithelial cell invasion by adherent-invasive Escherichia coli (AIEC) and other multidrug-resistant pathogens. The method addresses limitations of the traditional gentamicin protection assay, particularly in the context of antibiotic-resistant isolates from inflammatory bowel disease (IBD) patients.
Key Study Components
Area of Science
- Microbiology
- Cellular infection assays
- Pathogen diagnostics
Background
- Epithelial cell invasion is a hallmark of several enteric pathogens, including Salmonella, Shigella, and Vibrio.
- AIEC is an emerging pathogen implicated in IBD pathogenesis.
- AIEC strains lack classic virulence factors, complicating genetic identification.
- Phenotypic classification relies on cell invasion assays, traditionally using gentamicin.
Purpose of Study
- To develop a reliable assay for quantifying AIEC invasion in the context of rising antibiotic resistance.
- To provide a diagnostic tool for screening IBD patients for AIEC.
- To enable assessment of cell invasion by multidrug-resistant pathogens.
Methods Used
- Amikacin-based protection assay to kill extracellular bacteria while sparing intracellular bacteria.
- Quantification of invasion using colony-forming units (CFU) recovered post-infection.
- Calculation of percent invasion score for comparative analysis.
- Immunostaining and confocal microscopy for qualitative visualization of invaded epithelial cells.
Main Results
- The amikacin-based assay effectively quantifies and visualizes AIEC invasion of epithelial cells.
- Amikacin is effective against strains resistant to other aminoglycosides and remains extracellular to host cells.
- The assay provides both quantitative and qualitative data on bacterial invasiveness.
- It is suitable for use with multidrug-resistant isolates from IBD patients.
Conclusions
- The amikacin-based protection assay is a robust alternative to gentamicin-based methods for assessing epithelial cell invasion.
- This method enhances diagnostic capabilities for AIEC and other resistant pathogens in clinical and research settings.
- It serves as a proxy for evaluating pathogen virulence and pathogenicity.
Frequently Asked Questions
Executive Industry Relevance
Quantifying epithelial cell invasion by multidrug-resistant Escherichia coli is critical for target validation and mechanistic de-risking in infectious disease research. The amikacin protection assay enables reliable phenotypic classification and comparative analysis of invasive potential, especially in the context of antibiotic resistance. This capability supports translational continuity from discovery through preclinical pathogen characterization and portfolio triage.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables functional validation of bacterial invasion phenotypes in disease-relevant epithelial models.
- Supports mechanistic de-risking by distinguishing intracellular from extracellular pathogen populations.
- Facilitates comparative assessment of strain invasiveness for target prioritization.
Screening & Assay Development
- Provides a standardized, quantitative assay for evaluating cell invasion by antibiotic-resistant isolates.
- Delivers reproducible percent invasion scores using CFU quantification post-infection.
- Supports qualitative visualization of invaded cells via immunostaining and confocal microscopy.
Translational & Preclinical Research
- Aligns with translational biomarker strategies by linking invasion phenotype to pathogenicity in IBD and other diseases.
- Enables risk-adjusted advancement of candidate strains or interventions based on invasion metrics.
- Offers continuity from in vitro discovery to preclinical infection models.
Pipeline & Workflow Integration
The amikacin protection assay fits within the early discovery to preclinical continuum, supporting both hypothesis testing and downstream screening of multidrug-resistant pathogens.
- Discovery Biology: Provides robust quantification of invasion as a proxy for virulence and mechanistic insight.
- Screening: Standardizes evaluation of strain invasiveness for high-throughput or comparative workflows.
- Analytics: Generates quantitative percent invasion scores and qualitative imaging outputs for cross-condition analysis.
- Translational Research: Bridges in vitro invasion data to preclinical and clinical pathogen characterization.
- Enterprise Reuse: Adaptable for diverse enteric pathogens and multidrug-resistant isolates across research programs.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence in invasion phenotypes and reduces ambiguity in pathogen classification.
- Operational Value: Enhances assay reproducibility and scalability for both research and diagnostic applications.
- Strategic Value: Informs go/no-go decisions for pathogen-focused portfolios and supports capital-efficient screening.
- Portfolio Impact: Enables risk-adjusted prioritization of strains or interventions based on validated invasion metrics.
Implementation Considerations
- Requires expertise in cell culture infection models and quantitative microbiology.
- Needs access to amikacin, CFU plating infrastructure, and confocal microscopy for qualitative analysis.
- Demands cross-team standardization of assay conditions and readouts for reproducibility.
- Adaptable to various epithelial cell lines and multidrug-resistant pathogen isolates.
- Assay reliability may depend on antibiotic susceptibility profiles of clinical isolates.
Biopharma FAQs
Chapters in this video
0:00
Introduction
0:29
Preparation of Adherent-Invasive E. coli (AIEC) and Infection Media
2:43
Plating Input AIEC Inoculum for Pre-Infection Quantification
3:55
Infection of Caco-2 Epithelial Cell Monolayers with AIEC
5:03
Treatment with Amikacin and Recovery of Intracellular Bacteria
7:38
Results
8:49
Conclusion
