Data Sheet 1_Enhanced detection of animal-derived microbial hazards in forested catchments using high-volume ultrafiltration and amplicon-based microbial source tracking.pdf

IntroductionMonitoring of drinking water sources is an essential component of broader public health practise. However, routine water monitoring programmes that follow established methodological standards, such as low-volume grab sampling with standard filtration, have limitations in being representative. Particularly for protected source waters where (wildlife introduced) pathogens are in low concentration and are not evenly distributed. Microbial source tracking (MST) offers a promising approach to close this gap, enabling more precise identification of faecal contamination sources and their associated risk. However, as with other culture- and molecular-based approaches, the sensitivity of MST is constrained by sample capture methodology, limited by sample volume, timing, and randomness of grab sampling.
MethodsThis study investigated the application of a high-volume sample concentration method (EasyElute ultrafiltration) to enhance microbial recovery from source water. All evaluation was conducted alongside standard grab sampling and filtration methods. Post-concentration analyses combined traditional culture-based quantification of faecal indicator organisms (FIOs) and reference pathogens, with 16S rRNA amplicon MST to provide an integrated approach to surveillance of animal-derived microbial risks in forested water supply catchments.
ResultsThe results demonstrated that high-volume ultrafiltration enhanced bacterial recovery from source water samples, although turbidity was observed to limit overall efficiency, highlighting potential operational challenges. Comparative analysis demonstrated that amplicon-based MST produced consistent faecal source attribution across both standard and ultrafiltration methods, showing greater sensitivity at increasing volumes.
DiscussionThis study advances MST methodology by demonstrating the feasibility and added sensitivity achievable through high-volume, concentrated sample collection approaches. This is particularly relevant where water samples are expected to carry low microbial loads, ultimately offering a practical approach for improving faecal source tracking and risk assessment for water sources to protect public health in water supply catchments.

引言
饮用水水源监测是广义公共卫生实践的重要组成部分。然而，遵循既定方法学标准的常规水质监测方案（如采用标准过滤的低体积瞬时采样）在样本代表性方面存在局限，此类局限在受保护水源水体中尤为突出——这类水体中野生生物携带的病原体浓度极低且分布不均。微生物溯源（Microbial Source Tracking, MST）为填补这一空白提供了极具潜力的解决方案，可实现粪便污染源及其相关风险的精准识别。然而，与其他基于培养或分子生物学的检测方法类似，微生物溯源的灵敏度受样本采集方法制约，受制于采样体积、采样时机以及瞬时采样的随机性。

方法
本研究探讨了大体积样本浓缩方法（EasyElute超滤法）在提升水源水体微生物回收率方面的应用。所有评估均与标准瞬时采样及过滤方法同步开展。浓缩后的分析整合了基于传统培养的粪便指示生物（Faecal Indicator Organisms, FIOs）与参考病原体定量检测，以及16S rRNA扩增子微生物溯源技术，从而为森林供水集水区的动物源微生物风险监测提供了一体化方案。

结果
研究结果表明，尽管大体积超滤法提升了水源水样的细菌回收率，但浊度会限制整体回收率，凸显了该方法潜在的操作挑战。对比分析显示，基于扩增子的微生物溯源技术在标准采样法与超滤法下均能实现一致的粪便源归因，且随着采样体积提升展现出更高的检测灵敏度。

讨论
本研究通过证实大体积浓缩样本采集方法的可行性与更高灵敏度，推动了微生物溯源方法学的发展。这在预计微生物负载量较低的水样场景中尤为关键，最终可为改善粪便源追踪与水源风险评估提供实用方案，进而保障供水集水区的公共卫生安全。