DataSheet_1_Premise plumbing bacterial communities in four European cities and their association with Legionella.docx

IntroductionLegionella species are Gram negative, facultative, intracellular bacteria found in natural and engineered water systems. Understanding the bacterial interactions underlying the success of Legionella in aquatic environments could be beneficial for control.
Materials and methodsWe aimed to profile, by 16S rRNA gene amplicon sequencing (V3-V4), the bacterial communities in premise plumbing systems of buildings in four European cities (Copenhagen, Warsaw, Rome, Athens), and identify positive and negative associations of specific community members to culturable Legionella.
ResultsThe coarse taxonomic composition was similar across the four cities, but Copenhagen and Warsaw had richer, more diverse communities than Athens and Rome, with a greater number of city-specific amplicon sequence variants (ASVs). The cities had statistically significant differences in bacterial communities at the ASV level, with relatively few shared ASVs. Out of 5,128 ASVs, 73 were classified as Legionella, and one or more of these were detected in most samples from each city (88.1% overall). Interestingly, the relative abundance of Legionella ASVs did not correlate with Legionella culture status. Overall, 44.2% of samples were Legionella culture positive: 71.4% in Warsaw, 62.2% in Athens, 22.2% in Rome, and 15.2% in Copenhagen. 54 specific ASVs and 42 genera had significant positive or negative associations with culturable Legionella. Negative associations included Staphylococcus, Pseudomonas, and Acinetobacter. Positive associations included several Nitrospira ASVs and one classified as Nitrosomodaceae oc32, ASVs in the amoeba-associated genera Craurococcus-Caldovatus and Reyranella, and the predatory genus Bdellovibrio.
DiscussionSome of these associations are well supported by laboratory studies, but others are the opposite of what was expected. This highlights the difficulties in translating pure culture results to in complex real-life scenarios. However, these positive and negative associations held across the four cities, across multiple buildings and plumbing compartments. This is important because developing better control measures, including probiotic approaches, will require an understanding of ecological relationships that can be generalised across different engineered water systems.

引言
军团菌（Legionella）是一类革兰氏阴性兼性胞内细菌，广泛分布于自然与人工给水系统中。解析军团菌在水生环境中成功定植与繁衍的细菌互作机制，可为其防控工作提供理论参考。

材料与方法
本研究采用16S rRNA基因扩增子测序（V3-V4区）技术，对欧洲四座城市（哥本哈根、华沙、罗马、雅典）的建筑室内供水管道系统中的细菌群落进行表征，并鉴定与可培养军团菌存在显著正、负相关关系的特定群落成员。

结果
四座城市的细菌群落粗分类学组成整体相似，但哥本哈根与华沙的群落丰富度与多样性均高于雅典与罗马，且城市特异性扩增子序列变体（amplicon sequence variant, ASV）数量更多。在ASV水平上，各城市的细菌群落组成存在统计学显著性差异，共享ASV的数量相对较少。在总计5128个ASV中，有73个被归类为军团菌，且各城市的多数样本（整体占比88.1%）均可检出至少一种军团菌ASV。值得注意的是，军团菌ASV的相对丰度与军团菌培养检测结果并无相关性。整体而言，44.2%的样本呈军团菌培养阳性：其中华沙为71.4%，雅典为62.2%，罗马为22.2%，哥本哈根为15.2%。共有54个特异性ASV与42个菌属与可培养军团菌存在显著的正、负相关关系：负相关类群包括葡萄球菌属（Staphylococcus）、假单胞菌属（Pseudomonas）与不动杆菌属（Acinetobacter）；正相关类群则涵盖多个硝化螺菌属（Nitrospira）ASV、1个归类为亚硝化单胞菌科（Nitrosomodaceae）oc32的类群、与变形虫共生的Craurococcus-Caldovatus属和Reyranella属，以及捕食性菌属蛭弧菌属（Bdellovibrio）。

讨论
部分上述关联已得到实验室研究的充分验证，但另有部分关联与预期结果相悖，这凸显了将纯培养实验结果推演至复杂真实场景的难度。不过，这些正、负关联在四座城市、多栋建筑及不同管道隔间中均保持稳定。这一点至关重要，因为开发包括益生菌调控策略在内的更优防控手段，需要获取可在不同人工给水系统中推广应用的生态关系认知。