Institut Pasteur de la Guadeloupe, Unité TReD-Path, Les Abymes, Guadeloupe, France.
Institut de Systématique, Évolution, Biodiversité (ISYEB), Muséum National d'Histoire Naturelle, CNRS, Sorbonne Université, EPHE, Université des Antilles, Pointe-à-Pitre, Guadeloupe, France.
Sci Total Environ. 2023 Nov 20;900:165816. doi: 10.1016/j.scitotenv.2023.165816. Epub 2023 Jul 26.
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.
自由生活的阿米巴原虫（FLA）是无处不在的原生动物，主要存在于水生环境中。它们是已知的阿米巴耐药菌（ARB）的重要储存库和传播媒介，其中大多数对人类具有致病性。然而，与 FLA 相关的天然细菌微生物组在很大程度上仍然未知。在此，我们对从瓜德罗普岛娱乐水中分离的不同 FLA 物种的天然细菌微生物组进行了描述。在不同的放牧条件下，对澳大利亚纳氏虫（Naegleria australiensis）、纳氏虫 WTP3、副瓦氏虫（Paravahlkampfia ustiana）和瓦氏虫 AK-2007（异变形虫科）的单培养物进行了连续传代培养。使用 16S rRNA 基因宏条形码对水和阿米巴包囊的整个细菌微生物组进行了描述。通过质谱（MALDI-TOF MS）、常规 16S PCR 和圆盘扩散法（评估细菌抗生素耐药性）分析可培养的 ARB 亚群。透射电子显微镜用于定位 ARB 在阿米巴体内的位置。根据 alpha 和 beta 多样性分析，FLA 细菌微生物组与其栖息地的细菌微生物组有显著差异。虽然在水中检测到 Vogesella 和 Aquabacterium 属，但最常见的 ARB 属于假单胞菌属、博氏菌属和大肠杆菌/志贺氏菌属。不同的 FLA 物种与不同的细菌分类群表现出暂时和永久的关联，表明宿主特异性。这些关联取决于传代次数和放牧条件。此外，纳氏虫、瓦氏虫和副瓦氏虫包囊中自然携带不动杆菌属、大肠杆菌属、肠杆菌属、假单胞菌属和微杆菌属的有活力的细菌，所有这些细菌都对人类具有致病性。据我们所知，这是首次证明 Paravahlkampfia 和 Vahlkampfia 是水中致病性 ARB 的宿主。在全球范围内，这些 ARB 存在于耐药包囊中，这代表着潜在的健康风险。为了确保娱乐用水的持续安全，必须（i）定期监测阿米巴和 ARB，以及（ii）增进对阿米巴-细菌相互作用的了解，以制定更好的水管理方案。
