Abstract：
Free-living amoebae (FLA) are ubiquitous protozoa mainly found in aquatic environments. They are well-known reservoirs and vectors for the transmission of amoeba-resistant bacteria (ARB), most of which are pathogenic to humans. Yet, the natural bacterial microbiota associated with FLA remains largely unknown. Herein, we characterized the natural bacterial microbiota of different FLA species isolated from recreational waters in Guadeloupe. Monoxenic cultures of Naegleria australiensis, Naegleria sp. WTP3, Paravahlkampfia ustiana and Vahlkampfia sp. AK-2007 (Heterolobosea lineage) were cultivated under different grazing conditions, during successive passages. The whole bacterial microbiota of the waters and the amoebal cysts was characterized using 16S rRNA gene metabarcoding. The culturable subset of ARB was analyzed by mass spectrometry (MALDI-TOF MS), conventional 16S PCR, and disk diffusion method (to assess bacterial antibiotic resistance). Transmission electron microscopy was used to locate the ARB inside the amoebae. According to alpha and beta-diversity analyses, FLA bacterial microbiota were significantly different from the ones of their habitat. While Vogesella and Aquabacterium genera were detected in water, the most common ARB belonged to Pseudomonas, Bosea, and Escherichia/Shigella genera. The different FLA species showed both temporary and permanent associations with differentially bacterial taxa, suggesting host specificity. These associations depend on the number of passages and grazing conditions. Additionally, Naegleria, Vahlkampfia and Paravahlkampfia cysts were shown to naturally harbor viable bacteria of the Acinetobacter, Escherichia, Enterobacter, Pseudomonas and Microbacterium genera, all being pathogenic to humans. To our knowledge, this is the first time Paravahlkampfia and Vahlkampfia have been demonstrated as hosts of pathogenic ARB in water. Globally, the persistence of these ARB inside resistant cysts represents a potential health risk. To ensure the continued safety of recreational waters, it is crucial to (i) regularly control both the amoebae and their ARB and (ii) improve knowledge on amoebae-bacteria interactions to establish better water management protocols.
摘要：
自由生活的变形虫（FLA）是普遍存在的原生动物，主要存在于水生环境中。它们是众所周知的传播变形虫抗性细菌(ARB)的储库和载体,其中大多数对人类有致病性。然而，与FLA相关的天然细菌微生物群在很大程度上仍然未知。在这里，我们对从瓜德罗普岛的娱乐水域中分离出的不同FLA物种的天然细菌微生物群进行了表征。澳大利亚Naegleria的单氧培养物，Naegleriasp.WTP3,ParavahlkampfiaustianaandVahlkampfiasp.AK-2007（杂种谱系）在不同的放牧条件下栽培，在连续的通道中。使用16SrRNA基因meta编码表征了水域和变形虫囊肿的整个细菌微生物群。通过质谱（MALDI-TOFMS）分析ARB的可培养子集，常规16SPCR，和圆盘扩散法（评估细菌抗生素耐药性）。透射电子显微镜用于定位变形虫内部的ARB。根据阿尔法和贝塔多样性分析，FLA细菌微生物群与它们的栖息地明显不同。虽然在水中检测到Vogesella和Aquabterium属，最常见的ARB属于假单胞菌,博西阿,和埃希氏菌/志贺氏菌属。不同的FLA物种显示出与差异细菌分类群的临时和永久关联，提示宿主特异性。这些关联取决于通道的数量和放牧条件。此外,Naegleria,Vahlkampfia和Paravahlkampfia囊肿被证明可以自然地携带不动杆菌的活细菌，埃希氏菌,肠杆菌,假单胞菌和微杆菌属，都是对人类致病的。据我们所知,这是Paravahlkampfia和Vahlkampfia首次被证明是水中致病性ARB的宿主。全球范围内,这些ARB在耐药囊肿内的持续存在代表着潜在的健康风险.为确保康乐水域的持续安全,（i）定期控制变形虫及其ARB以及（ii）提高对变形虫-细菌相互作用的认识以建立更好的水管理方案至关重要。
