High Diversity and Prevalence of Potentially Pathogenic Free-Living Amoebae in Water Sources from Castilla y León, Spain

Abstract

Free-living amoebae (FLA) such as Acanthamoeba spp., Balamuthia mandrillaris, Naegleria fowleri, Sappinia pedata, Vermamoeba vermiformis and Vahlkampfia spp. are causal agents of deadly and/or disabling infections in humans. Despite recent data showing an increase in infection cases worldwide, studies on the prevalence of these emerging pathogens in water sources are scarce. Moreover, climate change is believed to facilitate the expansion and persistence of these environmental pathogens, further emphasizing the need for comprehensive surveillance. Therefore, the current study investigates the variety and abundance of free-living amoebae in different water sources in the autonomous community of Castilla y León, Spain, during different seasons of the year. Vermamoeba vermiformis was the most prevalent species and was detected in rivers, swamps, irrigation waters, swimming pools and recreational fountains. Moreover, genera such as Acanthamoeba and Naegleria and Vahlkampfia were also identified. This study highlights the diversity of FLA in the region and their relationship with local water characteristics. Given that certain FLA species are opportunistic pathogens, these results emphasize the necessity of monitoring this area and water sources.

Keywords: Acanthamoeba spp.; Castilla y León; Spain; Vermamoeba vermiformis; monitoring; water.

Figure 1

Geographical localization of the autonomous community of Castilla y León.

Figure 2

Distribution of free-living amoebae isolated in water samples for each province. The x-axis shows the provinces such as Salamanca, Valladolid, Zamora and Burgos. And the y-axis represents the number of positive samples and diversity of FLA species in the water samples. No FLA detected indicates the absence of free-living amoebae in the samples analyzed. Other FLA are amoebae not identified. Some of the samples contain more than one amoeba species.

Figure 3

Trophozoites and cysts of Naegleria americana from Salamanca’s river (SW23) for first sampling. Image was obtained with an ECHO Revolution hybrid microscope (40×). Scale bar represents 100 μM.

Figure 4

Trophozoites of Vannella spp. in a recreational fountain from Valladolid (VW5) for first sampling. Image was obtained with an ECHO Revolution hybrid microscope (40×). Scale bar represents 100 μM.

Figure 5

Trophozoites of unidentified amoeba from a recreational fountain sample from Valladolid VW5 (A) and Zamora ZW4 (B) for third sampling. Image was obtained with an ECHO Revolution hybrid microscope (40×). Scale bar represents 100 μM.

Figure 6

Cyst of Vermamoeba vermiformis (A) and trophozoites and cyst of Acanthamoeba spp. (B). Image was obtained with a LEICA DM500 microscope (20×). Scale bar represents 100 μM.

Figure 7

Distribution of free-living amoebae isolated in water samples for each season. The x-axis shows the season (t1–t3). And the y-axis represents the prevalence and diversity of FLA in the water samples. No FLA detected indicates the absence of free-living amoebae in the samples analyzed. Other FLA are amoebae not identified.

Figure 8

Distribution of free-living amoebae isolated in water samples for type of water. The x-axis shows the different types of water samples collected in different provinces such as Salamanca, Valladolid, Zamora, and Burgos. And the y-axis represents the number of positive samples and diversity of FLA species in the water samples. No FLA detected indicates the absence of free-living amoebae in the samples analyzed. Other FLA are amoebae not identified. Some of the samples contain more than one amoeba species.

Figure 9

Phylogenetic connections among the species Vermamoeba vermiformis obtained from water samples in this study. The isolates obtained in this study are marked within boxes. Other species sequences were obtained from GenBank and their accession numbers are shown. The tree is computed using the Maximum Likelihood method and measured in the number of substitutions per site. The tree is rooted with Balamuthia mandrillaris as the outgroup. The percentage of replicate trees, in which the associated taxa are clustered together in the bootstrap test, is shown next to the branches (in bold). Scale bar = 2.00 substitutions/site.

Figure 10

Phylogenetic connections among the family Vahlkampfiidae obtained from water samples in this study. The isolates obtained in this study are marked within boxes. Other species sequences were obtained from GenBank and their accession numbers are shown. The tree is computed using the Maximum Likelihood method and measured in the number of substitutions per site. The tree is rooted with Vermamoeba vermiformis as the outgroup. The percentage of replicate trees, in which the associated taxa are clustered together in the bootstrap test, is shown next to the branches (in bold). Scale bar = 0.20 substitutions/site.

Figure 11

Phylogenetic connections among the genus Acanthamoeba obtained from water samples in this study. The isolates obtained in this study are marked within boxes. Other species sequences were obtained from GenBank and their accession numbers are shown. The tree is computed using the Maximum Likelihood method and measured in the number of substitutions per site. The tree is rooted with Naegleria fowleri as the outgroup. The percentage of replicate trees, in which the associated taxa are clustered together in the bootstrap test, is shown next to the branches (in bold). Scale bar = 0.20 substitutions/site.