Abstract：
Free-living amoebae (FLA) such as Acanthamoeba, Balamuthia mandrillaris, Naegleria fowleri and Sappinia pedata are naturally widespread in freshwater, causing rare but fatal and debilitating infections in humans. Although recent studies have shown an increase in infection rates, there is a paucity of epidemiological studies regarding the presence of these emerging pathogens in water. Herein, we studied the diversity and relative abundance of thermophilic FLA in different recreational baths in a tropical climate for 5 years. From 2018 to 2022, a total of 96 water samples were collected from 7 recreational baths (natural, tiled, regularly cleaned or not, and with temperatures ranging from 27 to 40 °C). DNA was extracted from FLA cultivated at 37 °C to detect thermophilic culturable FLA. Metabarcoding studies were conducted through FLA 18S rRNA gene amplicons sequencing; amplicon sequence variants (ASV) were extracted from each sample and taxonomy assigned against PR2 database using dada2 and phyloseq tools. We also searched for Naegleria sp. and N. fowleri using PCR targeting ITS and NFITS genes (respectively) and we quantified them using an optimized most probable number (MPN) method for FLA. Our results showed that differences in FLA diversity and abundance were observed amongst the 7 baths, but without a clear seasonal distribution. Naegleria, Vermamoeba and Stenamoeba were the most represented genera, while the genera Acanthamoeba and Vahlkampfia were mainly found in 2 baths. The MPN values for Naegleria sp. (NT/l) increased between 2018 and 2022, but the MPN values for N. fowleri (NF/l) seemed to decrease. Globally, our results showed that since we cannot establish a seasonal distribution of FLA, the regular presence of FLA (namely Naegleria and Acanthamoeba) in recreational waters can pose a potential threat in terms of neuroinfections as well as Acanthamoeba keratitis. It is thus imperious to perform the regular control of these baths as a preventive health measure.
摘要：
自由生活的变形虫（FLA），如棘阿米巴，Balamuthiamandrillaris,Naegleriafowleri和Sappinia在淡水中自然分布，在人类中引起罕见但致命和衰弱的感染。尽管最近的研究表明感染率上升，关于水中这些新出现的病原体的流行病学研究很少。在这里，我们研究了5年热带气候下不同休闲浴场中嗜热FLA的多样性和相对丰度。从2018年到2022年，共收集了7个休闲浴场的96个水样(自然，平铺,定期清洁或不清洁，温度范围为27至40°C）。从37°C培养的FLA中提取DNA以检测嗜热可培养FLA。通过FLA18SrDNA扩增子测序进行了元编码研究;从每个样品中提取扩增子序列变体（ASV），并使用dada2和phyloseq工具针对PR2数据库分配分类法。我们还使用针对ITS和NFITS的PCR（分别）搜索了Naegleria和N.fowleri，并使用FLA的优化的最可能数（MPN）方法对其进行了定量。我们的结果表明，在7个浴场中观察到FLA多样性和丰度的差异，但没有明确的季节分布。Naegleria,Vermamoeba和Stenamoeba是最具代表性的属，而棘阿米巴属和Vahlkampfia属主要分布在2个浴场中。Naegleriasp（NT/L）的MPN值在2018年至2022年之间增加，但N.fowleri（NF/L）的MPN值似乎减少。全球范围内,我们的结果表明，由于我们不能建立FLA的季节性分布，在休闲水域中经常存在FLA（即Naegleria和棘阿米巴）可能会对神经感染和棘阿米巴角膜炎构成潜在威胁。因此，作为预防性健康措施，必须定期控制这些浴池。
