Lebeuf Maria, Turgeon Nathalie, Faubert Cynthia, Pleau Alexandre, Robillard Justin, Paradis Éric, Marette André, Duchaine Caroline
Département de Biochimie, Microbiologie et Bio-Informatique, Université Laval, Quebec City, QC, Canada.
Institut Universitaire de Cardiologie et de Pneumologie de Québec, Quebec City, QC, Canada.
Front Microbiol. 2021 Aug 16;12:709399. doi: 10.3389/fmicb.2021.709399. eCollection 2021.
The use of axenic animal models in experimental research has exponentially grown in the past few years and the most reliable way for confirming their axenic status remains unclear. It is especially the case when using individual ventilated positive-pressure cages such as the Isocage. This type of cage are at a greater risk of contamination and expose animals to a longer handling process leading to more potential stress when opened compared to isolators. The aim of this study was to propose simple ways to detect microbial contaminants with Isocages type isolator resulting by developing, validating and optimizing three different methods (culture, microscopy, and molecular). These three approaches were also tested by spiking 21 axenic mice with different microorganisms. Our results suggest that the culture method can be used for feces and surface station (IBS) swabs exclusively (in Brain Heart Infusion for 7 days at 25°C and 37°C in aerobic conditions, and at 30°C in anaerobic conditions), while microscopy (wet mounts) and molecular method (quantitative PCR) were only suitable for fecal matter analyses. results suggests that the culture and molecular methods can detect up to 100% of bacterial contamination events while the microscopy approach generates many erroneous results when not performed by a skilled microscopist. results also suggest that when an axenic mouse is contaminated by a microbial agent, the microorganism will colonize the mouse to such an extent that detection is obvious in 4 days, in average. This report validates simple but complimentary tests that can be used for optimal detection of contaminants in axenic animal facilities using Isocage type isolators.
在过去几年中，无菌动物模型在实验研究中的应用呈指数级增长，而确认其无菌状态的最可靠方法仍不明确。使用诸如Isocage等个体通气正压笼时尤其如此。与隔离器相比，这种类型的笼子污染风险更大，且动物在打开笼子时会经历更长的处理过程，从而导致更多潜在压力。本研究的目的是通过开发、验证和优化三种不同方法（培养、显微镜检查和分子方法），提出检测Isocage型隔离器产生的微生物污染物的简单方法。这三种方法还通过向21只无菌小鼠接种不同微生物进行了测试。我们的结果表明，培养方法仅可用于粪便和表面站（IBS）拭子（在脑心浸液中，于25°C和37°C有氧条件下培养7天，在30°C厌氧条件下培养），而显微镜检查（湿片法）和分子方法（定量PCR）仅适用于粪便分析。结果表明，培养和分子方法可检测高达100%的细菌污染事件，而显微镜检查方法若由非熟练显微镜操作人员进行，则会产生许多错误结果。结果还表明，当无菌小鼠被微生物制剂污染时，该微生物会在小鼠体内定殖，平均4天内检测结果就会很明显。本报告验证了可用于使用Isocage型隔离器的无菌动物设施中污染物最佳检测的简单但互补的测试方法。
