Free-living amoebae (FLA) are prevalent in nature and man-made environments, and they can survive in harsh conditions by forming cysts. Studies have discovered that some FLA species are able to show pathogenicity to human health, leading to severe infections of central nervous systems, eyes, etc. with an extremely low rate of recovery. Therefore, it is imperative to establish a surveillance framework for FLA in environmental habitats. While many studies investigated the risks of independent FLA, interactions between FLA and surrounding microorganisms determined microbial communities in ecosystems and further largely influenced public health. Here we systematically discussed the interactions between FLA and different types of microorganisms and corresponding influences on behaviors and health risks of FLA in the environment. Specifically, bacteria, viruses, and eukaryotes can interact with FLA and cause either enhanced or inhibited effects on FLA infectivity, along with microorganism community changes. Therefore, considering the co-existence of FLA and other microorganisms in the environment is of great importance for reducing environmental health risks.

Legionella is the causative agent of Legionnaires\' disease, and its prevalence in potable water is a significant public health issue. Water stagnation within buildings increases the risk of Legionella. However, there are limited studies investigating how stagnation arising through intermittent usage affects Legionella proliferation and the studies that are available do not consider viable but non culturable (VBNC) Legionella. This study used a model plumbing system to examine how intermittent water stagnation affects both VBNC and culturable Legionella. The model plumbing system contained a water tank supplying two biofilm reactors. The model was initially left stagnant for ≈5 months (147 days), after which one reactor was flushed daily, and the other weekly. Biofilm coupons, and water samples were collected for analysis at days 0, 14 and 28. These samples were analysed for culturable and VBNC Legionella, free-living amoebae, and heterotrophic bacteria. After 28 days, once-a-day flushing significantly (p < 0.001) reduced the amount of biofilm-associated culturable Legionella (1.5 log10 reduction) compared with weekly flushing. However, higher counts of biofilm-associated VBNC Legionella (1 log10 higher) were recovered from the reactor with once-a-day flushing compared with weekly flushing. Likewise, once-a-day flushing increased the population of biofilm-associated Vermamoeba vermiformis (approximately 3 log10 higher) compared with weekly flushing, which indicated a positive relationship between VBNC Legionella and V. vermiformis. This is the first study to investigate the influence of stagnation on VBNC Legionella under environmental conditions. Overall, this study showed that a reduction in water stagnation decreased culturable Legionella but not VBNC Legionella.

Parasites and free-living amoebae (FLA) are common pathogens that pose threats to wildlife and humans. The black-necked crane (Grus nigricollis) is a near-threatened species and there is a shortage of research on its parasite diversity. Our study aimed to use noninvasive methods to detect intestinal parasites and pathogenic FLA in G. nigricollis using high-throughput sequencing (HTS) based on the 18S rDNA V9 region. A total of 38 fresh fecal samples were collected in Dashanbao, China, during the overwintering period (early-, middle I-, middle II-, and late-winter). Based on the 18S data, eight genera of parasites were identified, including three protozoan parasites: Eimeria sp. (92.1%) was the dominant parasite, followed by Tetratrichomonas sp. (36.8%) and Theileria sp. (2.6%). Five genera of helminths were found: Echinostoma sp. (100%), Posthodiplostomum sp. (50.0%), Euryhelmis sp. (26.3%), Eucoleus sp. (50.0%), and Halomonhystera sp. (2.6%). Additionally, eight genera of FLA were detected, including the known pathogens Acanthamoeba spp. (n = 13) and Allovahlkampfia spp. (n = 3). Specific PCRs were used to further identify the species of some parasites and FLA. Furthermore, the 18S data indicated significant changes in the relative abundance and genus diversity of the protozoan parasites and FLA among the four periods. These results underscore the importance of long-term monitoring of pathogens in black-necked cranes to protect this near-endangered species.
UNASSIGNED: Métabarcoding des protozoaires et des helminthes chez les grues à cou noir : forte prévalence de parasites et d’amibes libres.
UNASSIGNED: Les parasites et les amibes libres sont des agents pathogènes courants qui constituent une menace pour la faune et les humains. La grue à cou noir (Grus nigricollis) est une espèce quasi menacée et les recherches sur sa diversité parasitaire sont insuffisantes. Notre étude visait à utiliser des méthodes non invasives pour détecter les parasites intestinaux et les amibes libres pathogènes chez G. nigricollis en utilisant le séquençage à haut débit basé sur la région V9 de l’ADNr 18S. Au total, 38 échantillons de matières fécales fraîches ont été collectés à Dashanbao, en Chine, au cours de la période d’hivernage (début, milieu I, milieu II et fin de l’hiver). Sur la base des données 18S, huit genres de parasites ont été identifiés, dont trois parasites protozoaires : Eimeria sp. (92,1 %) était le parasite dominant, suivi de Tetratrichomonas sp. (36,8 %) et Theileria sp. (2,6 %). Cinq genres d’helminthes ont été trouvés : Echinostoma sp. (100 %), Posthodiplostomum sp. (50,0 %), Euryhelmis sp. (26,3 %), Eucoleus sp. (50,0 %) et Halomonhystera sp. (2,6 %). De plus, huit genres d’amibes libres ont été détectés, y compris les agents pathogènes connus Acanthamoeba spp. (n = 13) et Allovahlkampfia spp. (n = 3). Des PCR spécifiques ont été utilisées pour identifier davantage les espèces de certains parasites et amibes libres. En outre, les données 18S ont indiqué des changements significatifs dans l’abondance relative et la diversité des genres des parasites protozoaires et des amibes au cours des quatre périodes. Ces résultats soulignent l’importance de la surveillance à long terme des agents pathogènes chez les grues à cou noir pour protéger cette espèce quasi menacée.

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In response to growing global interest in organic agriculture, this study delves into the microbial landscape of organically grown raw produce with a focus on food safety. Vegetables that are consumed raw are potential vehicles for the transmission of any type of microorganism capable of causing human disease. Free-living amoebae (FLA) are ubiquitous protozoa found in many ecosystems and can serve as hosts to pathogenic bacteria. So far, data regarding the FLA bacterial microbiome in fresh produce remain scarce and are non-existent for those of organic origin. Thus, the aim of this preliminary work is to characterize the microbiome of FLA in commonly consumed raw vegetables to know their possible implications for consumers. A total of 40 organic cabbage, lettuce, spinach, and strawberry samples were analyzed. FLA were found in all samples, and their bacterial microbiome was obtained via amplicon sequencing using the Illumina MiSeq platform and pair-end protocol. Acanthamoeba spp. and Vermamoeba vermiformis were identified via qPCR in 65.0% and 25.0% of the samples, respectively. Regarding the bacterial microbiome of FLA, the most abundant genera were Pseudomonas (1.8-17.8%) and Flavobacterium (1.7-12.6%). Bacteria not previously related to FLA, such as Prosthecobacter or Cellvibrio, are described in this work. Importantly, several bacterial genera found within the FLA microbiome were identified as potential human pathogens, including Pseudomonas, Flavobacterium, Arcobacter, Klebsiella, Mycobacterium, Salmonella and Legionella. This is the first work in which FLA microbiome isolated from organic products has been characterized, underscoring the significance of understanding FLA\'s role as carriers of pathogenic bacteria in the context of organic food safety.

In this study, the presence of Acanthamoeba spp., Blastocystis sp., Cryptosporidium spp., Cyclospora cayetanensis, Entamoeba histolytica, Giardia sp., Toxoplasma gondii and Vermamoeba vermiformis was assessed in organic leafy green vegetables (lettuce, spinach, cabbage) and fruits (strawberry), which are usually consumed raw. A total of 110 organic samples were collected in Valencia (Spain). Protozoa were concentrated before detection by immunofluorescence (Cryptosporidium spp. and Giardia sp.) or real-time qPCR (Acanthamoeba spp., Blastocystis sp., C. cayetanensis, E. histolytica, T. gondii and V. vermiformis). The most abundant protozoa in organic vegetables and berry fruits were Acanthamoeba (65.5%), followed by T. gondii (37.2%), V. vermiformis (17.3%), C. cayetanensis (12.7%), Cryptosporidium spp. (6.8%), Blastocystis sp. (1.8%) and Giardia sp. (1.7%). E. histolytica was not found in any of the organic samples. Thus, results showed that consumers can be exposed to protozoan parasites by consuming organic vegetables and berry fruits. This is the first report in Spain describing the presence of the protozoan pathogens Acanthamoeba spp., Blastocystis sp., C. cayetanensis, T. gondii and V. vermiformis, Cryptosporidium spp. and Giardia sp. in organic fresh produce. The results of this research will help determine the risk of foodborne protozoan parasites on organic leafy greens and strawberries that are available at local markets.

Hospital water systems are a significant source of Legionella, resulting in the potentially fatal Legionnaires\' disease. One of the biggest challenges for Legionella management within these systems is that under unfavorable conditions Legionella transforms itself into a viable but non culturable (VBNC) state that cannot be detected using the standard methods. This study used a novel method (flow cytometry-cell sorting and qPCR [VFC+qPCR] assay) concurrently with the standard detection methods to examine the effect of temporary water stagnation, on Legionella spp. and microbial communities present in a hospital water system. Water samples were also analyzed for amoebae using culture and Vermamoeba vermiformis and Acanthamoeba specific qPCR. The water temperature, number and duration of water flow events for the hand basins and showers sampled was measured using the Enware Smart Flow® monitoring system. qPCR analysis demonstrated that 21.8% samples were positive for Legionella spp., 21% for L. pneumophila, 40.9% for V. vermiformis and 4.2% for Acanthamoeba. All samples that were Legionella spp. positive using qPCR (22%) were also positive for VBNC Legionella spp.; however, only 2.5% of samples were positive for culturable Legionella spp. 18.1% of the samples were positive for free-living amoebae (FLA) using culture. All samples positive for Legionella spp. were also positive for FLA. Samples with a high heterotrophic plate count (HPC ≥ 5 × 103 CFU/L) were also significantly associated with high concentrations of Legionella spp. DNA, VBNC Legionella spp./L. pneumophila (p < 0.01) and V. vermiformis (p < 0.05). Temporary water stagnation arising through intermittent usage (< 2 hours of usage per month) significantly (p < 0.01) increased the amount of Legionella spp. DNA, VBNC Legionella spp./L. pneumophila, and V. vermiformis; however, it did not significantly impact the HPC load. In contrast to stagnation, no relationship was observed between the microbes and water temperature. In conclusion, Legionella spp. (DNA and VBNC) was associated with V. vermiformis, heterotrophic bacteria, and stagnation occurring through intermittent usage. This is the first study to monitor VBNC Legionella spp. within a hospital water system. The high percentage of false negative Legionella spp. results provided by the culture method supports the use of either qPCR or VFC+qPCR to monitor Legionella spp. contamination within hospital water systems.

Free-living amoebae (FLA) are phagocytic protists that play crucial roles in microbial communities as significant microbial grazers. However, our current knowledge of their diversity, ecology, and population genetic structures is marginal due to the shallow and biased sampling of ecosystems and the use of few, poorly resolving molecular markers. Thirty-two FLA were isolated from soil and water samples collected across representative ecosystems of the State of Morelos in Central Mexico, including the drinking water distribution system (DWDS) from the state capital. We classified our isolates as members of Acanthamoeba, Vermamoeba, Naegleria, and Tetramitus by 18S ribosomal DNA (rDNA) sequencing. Vermamoeba isolates were recovered exclusively from the DWDS samples. In contrast, Naegleria strains displayed a broad distribution in soil and water samples across the natural ecosystems. We used a combination of phylogenetic and population genetic analyses of internal transcribed spacer (ITS) and cytochrome oxidase subunit I (COI) sequences from our isolates and a comprehensive set of reference sequences to analyze the currently known diversity of Naegleria spp. Significant associations were uncovered between the most prevalent lineages of Naegleria and Vermamoeba and broad ecological and geographical variables at regional and global levels. The population structure and cryptic diversity within the Naegleria galeacystis-Naegleria americana and Vermamoeba vermiformis species complexes were thoroughly analyzed. Our results prove that the genus Vermamoeba, which was previously thought to consist of only one species, actually encompasses at least seven widely distributed species, as indicated by consistent evidence from Bayesian phylogenetics, two species-delimitation programs, and population genetics analyses. IMPORTANCE Our study sheds new light on the population genetic structure of V. vermiformis and diverse Naegleria species. Using improved molecular markers and advanced analytical approaches, we discovered that N. americana, previously considered a single species, actually contains multiple distinct lineages, as revealed by COI sequencing. These lineages are highly differentiated, with little gene flow between them. Our findings demonstrate that the genus Vermamoeba holds multiple cryptic species, requiring a significant taxonomic revision in light of multilocus sequence analyses. These results advance our understanding of the ecology, molecular systematics, and biogeography of these genera and species complexes at both regional and global scales. This study has significant implications for diagnosing amoebal infections and evaluating health risks associated with FLA in domestic and recreational waters.

UNASSIGNED: Free-living amoebae of the Naegleria genus belong to the major protist clade Heterolobosea and are ubiquitously distributed in soil and freshwater habitats. Of the 47 Naegleria species described, N. fowleri is the only one being pathogenic to humans, causing a rare but fulminant primary amoebic meningoencephalitis. Some Naegleria genome sequences are publicly available, but the genetic basis for Naegleria diversity and ability to thrive in diverse environments (including human brain) remains unclear.
UNASSIGNED: Herein, we constructed a high-quality Naegleria genus pangenome to obtain a comprehensive catalog of genes encoded by these amoebae. For this, we first sequenced, assembled, and annotated six new Naegleria genomes.
UNASSIGNED: Genome architecture analyses revealed that Naegleria may use genome plasticity features such as ploidy/aneuploidy to modulate their behavior in different environments. When comparing 14 near-to-complete genome sequences, our results estimated the theoretical Naegleria pangenome as a closed genome, with 13,943 genes, including 3,563 core and 10,380 accessory genes. The functional annotations revealed that a large fraction of Naegleria genes show significant sequence similarity with those already described in other kingdoms, namely Animalia and Plantae. Comparative analyses highlighted a remarkable genomic heterogeneity, even for closely related strains and demonstrate that Naegleria harbors extensive genome variability, reflected in different metabolic repertoires. If Naegleria core genome was enriched in conserved genes essential for metabolic, regulatory and survival processes, the accessory genome revealed the presence of genes involved in stress response, macromolecule modifications, cell signaling and immune response. Commonly reported N. fowleri virulence-associated genes were present in both core and accessory genomes, suggesting that N. fowleri\'s ability to infect human brain could be related to its unique species-specific genes (mostly of unknown function) and/or to differential gene expression. The construction of Naegleria first pangenome allowed us to move away from a single reference genome (that does not necessarily represent each species as a whole) and to identify essential and dispensable genes in Naegleria evolution, diversity and biology, paving the way for further genomic and post-genomic studies.

Legionellosis, an often-lethal pneumonia, is generally associated with contamination by Legionella pneumophila. This bacterium can persist in the environment and resist chemical treatment when it is internalized by amoebae. In addition, there is increasing medical proof that other Legionella species can be causative agents of Legionellosis. The objective of this study was to evaluate whether Legionella non-pneumophila (Lnp) strains were able to use the machinery of amoeba to multiply, or whether amoebae were able to limit their proliferation. Seven strains belonging to the species L. longbeachae, L. anisa, L. bozemanae, L. taurinensis, and L. dumoffii were cocultured with three amoebae, Acanthamoeba castellanii, Willaertia magna T5(S)44, and Willaertia magna C2c Maky, at two temperatures, 22 and 37 °C. We found that at 22 °C, all amoebae were able to phagocytose the seven Lnp strains and to avoid intracellular development, except for L. longbeachae, which was able to multiply inside W. magna T5(S)44. At 37 °C, four Lnp strains were able to hijack the machinery of one or two amoebae and to use it to proliferate, but none were able to multiply inside W. magna C2c Maky.