Isolation and Molecular Identification of Vahlkampfiidae and Vermamoeba Vermiformis from Fresh Vegetables: A Neglected Source of Infections.

Background: Naegleria spp., Tetramitus spp., and Vermamoeba vermiformis are potential pathogenic free-living amoebae (FLA) causing diseases such as keratitis, meningoencephalitis, and lung infections. We aimed to investigate the presence of Vahlkampfiidae and V. vermiformis in raw vegetables commonly consumed in Iran. Methods: Totally, 70 samples of vegetables samples including watercress (22), leeks (12), parsley (10), basil (13) and mint (13) were collected from municipal markets of Tehran, the capital of Iran during June to October 2021. After washing vegetables, samples were cultivated onto 2% non-nutrient agar (NNA) medium. After morphological confirmations, DNA was extracted and identical fragments of the FLA were amplified and sequenced. Results: Out of 70 cultured samples, 11 samples (15.71 %) were morphologically positive, of which four and seven were V. vermiformis and Vahlkampfiidae isolates, respectively. According to the PCR/sequencing results two, one, one, and one strains belonged to N. australiensis, N. americana, Vahlkampfia sp., V. inornata, and T. aberdonicus, respectively. All Vermamoeba genus were characterized as V. vermiformis. Conclusion: The results of current study revealed the contamination of fresh raw vegetables with Vahlkampfiidae and V. vermiformis. In addition, to our knowledge this is the first report of T. aberdonicus in raw vegetables. Our findings highlight the public health importance of vegetables in transmission of FLA, as well as the potential role of FLA in transmission of potential pathogenic microorganisms via consuming of fresh raw vegetables.

Mass mortality of Cornu aspersum in Italian snail farms: A histopathological survey.

The production demand of edible snails in the Mediterranean area is very high and the attention to snail borne diseases is increasing. Following mass mortality events, we have analyzed 240 samples of Cornu aspersum collected from farms across Italy. Anatomopathological examination showed the presence of alterations of the gastro-intestinal apparatus and of the digestive gland, while histopathological examination revealed the presence of Rickettsia-like organisms (RLOs) in 70% (168/240) of cases and Giemsa positive amoebae in the remaining 30% (72/240) of cases. RLOs were localized mainly at the level of the DG, where regressive changes or nodular inflammation was observed. TEM examination of RLOs samples revealed the presence of many rod-shaped electron dense microorganisms. Amoebal infection occurred in the kidney, intestine, lung, the DG and were associated to regressive events or infiltrative/nodular and encapsulation like inflammation. To date it is still unclear if the pathogens detected could represent a risk for humans and animals, therefore further studies are needed to better elucidate this point.

Sodium Metabisulfite Inhibits Acanthamoeba Trophozoite Growth through Thiamine Depletion.

Acanthamoeba keratitis (AK) is a severe infection of the cornea. Prevention and treatment are difficult due to the inefficacy of currently available compounds. The impact of many commonly used compounds for routine examinations of Acanthamoeba is unexplored but might offer insight useful in combatting AK. In this study, we demonstrate that sodium metabisulfite, a common preservation constituent of eye care solutions, was found to be active against Acanthamoeba trophozoites at concentrations lower than that commonly found in eye drops (IC50 0.03 mg/mL). We demonstrate that sodium metabisulfite depletes thiamine from growth medium and that Acanthamoeba is a thiamine auxotroph, requiring thiamine salvage for growth. The inhibitory effects of sodium metabisulfite can be overcome by thiamine supplementation. These results are consistent with the lack of key enzymes for thiamine biosynthesis in the genome of Acanthamoeba, an area which might prove exploitable using new or existing compounds. Indeed, this study highlights sodium metabisulfite as a useful inhibitor of Acanthamoeba castellanii trophozoites in vitro and that it acts, at least in part, by limiting available thiamine.

Nano- and microplastics drive the dynamic equilibrium of amoeba-associated bacteria and antibiotic resistance genes.

As emerging pollutants, microplastics have become pervasive on a global scale, inflicting significant harm upon ecosystems. However, the impact of these microplastics on the symbiotic relationship between protists and bacteria remains poorly understood. In this study, we investigated the mechanisms through which nano- and microplastics of varying sizes and concentrations influence the amoeba-bacterial symbiotic system. The findings reveal that nano- and microplastics exert deleterious effects on the adaptability of the amoeba host, with the magnitude of these effects contingent upon particle size and concentration. Furthermore, nano- and microplastics disrupt the initial equilibrium in the symbiotic relationship between amoeba and bacteria, with nano-plastics demonstrating a reduced ability to colonize symbiotic bacteria within the amoeba host when compared to their microplastic counterparts. Moreover, nano- and microplastics enhance the relative abundance of antibiotic resistance genes and heavy metal resistance genes in the bacteria residing within the amoeba host, which undoubtedly increases the potential transmission risk of both human pathogens and resistance genes within the environment. In sum, the results presented herein provide a novel perspective and theoretical foundation for the study of interactions between microplastics and microbial symbiotic systems, along with the establishment of risk assessment systems for ecological environments and human health.

Presence and diversity of free-living amoebae and their potential application as water quality indicators.

Free-living amoebae (FLA) are found in diverse environments, such as soils, rivers, and seas. Hence, they can be used as bioindicators to assess the water quality based solely on their presence. In this study, we determined the presence of FLA in river water by filtering water samples collected from various sites and culturing the resulting filtrates. FLA were detected in all the water samples with varying quality grades (Grades Ι-V). The significant increase in the size of the amoebae population with the deterioration in the water quality. Monoxenic cultures of the amoebae were performed, and genomic DNAs were isolated, among which 18S rDNAs were sequenced to identify the amoeba species. Of the 12 species identified, 10 belonged to the Acanthamoeba genus; of the remaining 2 species, one was identified as Vannella croatica and the other as a species of Vermamoeba. Acanthamoeba was detected in samples with Grades Ι to VI quality, whereas the Vermamoeba species was present only in Grade Ι water. V. croatica was found exclusively in water with Grade ΙΙ quality. Following morphological observations, genomic DNA was sequenced using 16S rDNA to determine whether the species of Acanthamoeba harbored endosymbionts. Most of the isolated Acanthamoeba contained endosymbionts, among which 4 species of endogenous bacteria were identified and examined using transmission electron microscopy. This study provides evidence that the distribution of amoebae other than Acanthamoeba may be associated with water quality. However, further confirmation will be required based on accurate water quality ratings and assessments using a more diverse range of FLA.

Exploring the extrachromosomal plasmid rDNA of Naegleria fowleri AY27 genotype II: A human brain-eating amoeba via high-throughput sequencing.

Naegleria fowleri, also known as brain-earing amoeba, causes severe and rapidly fatal CNS infection in humans called primary amebic meningoencephalitis (PAM). The DNA from the N. fowleri clinical isolate was sequenced for circular extrachromosomal ribosomal DNA (CERE - rDNA). The CERE contains 18 S, 5.8 S, and 28 S ribosomal subunits separated by internal transcribed spacers, 5 open reading frames (ORFs), and mostly repeat elements comprising 7268 bp out of 15,786 bp (46%). A wide variety of variations and recombination events were observed. Finally, the ORFs that comprised only 4 hypothetical proteins were modeled and screened against Zinc drug-like compounds. Two compounds [ZINC77564275 (ethyl 2-(((4-isopropyl-4 H-1,2,4-triazol-3-yl) methyl) (methyl)amino) oxazole-4-carboxylate) and ZINC15022129 (5-(2-methoxyphenoxy)-[2,2'-bipyrimidine]-4,6(1 H,5 H)-dione)] were finalized as potential druggable compounds based on ADME toxicity analysis. We propose that the compounds showing the least toxicity would be potential drug candidates after laboratory experimental validation is performed.

The isoprenyl chain length of coenzyme Q mediates the nutritional resistance of fungi to amoeba predation.

Amoebae are environmental predators feeding on bacteria, fungi, and other eukaryotic microbes. Predatory interactions alter microbial communities and impose selective pressure toward phagocytic resistance or escape which may, in turn, foster virulence attributes. The ubiquitous fungivorous amoeba Protostelium aurantium has a wide prey spectrum in the fungal kingdom but discriminates against members of the Saccharomyces clade, such as Saccharomyces cerevisiae and Candida glabrata. Here, we show that this prey discrimination among fungi is solely based on the presence of ubiquinone as an essential cofactor for the predator. While the amoeba readily fed on fungi with CoQ presenting longer isoprenyl side chain variants CoQ8-10, such as those from the Candida clade, it failed to proliferate on those with shorter CoQ variants, specifically from the Saccharomyces clade (CoQ6). Supplementing non-edible yeast with CoQ9 or CoQ10 rescued the growth of P. aurantium, highlighting the importance of a long isoprenyl side chain. Heterologous biosynthesis of CoQ9 in S. cerevisiae by introducing genes responsible for CoQ9 production from the evolutionary more basic Yarrowia lipolytica complemented the function of the native CoQ6. The results suggest that the use of CoQ6 among members of the Saccharomyces clade might have originated as a predatory escape strategy in fungal lineages and could be retained in organisms that were able to thrive by fermentation. IMPORTANCE: Ubiquinones (CoQ) are universal electron carriers in the respiratory chain of all aerobic bacteria and eukaryotes. Usually 8-10 isoprenyl units ensure their localization within the lipid bilayer. Members of the Saccharomyces clade among fungi are unique in using only 6. The reason for this is unclear. Here we provide evidence that the use of CoQ6 efficiently protects these fungi from predation by the ubiquitous fungivorous amoeba Protostelium aurantium which lacks its own biosynthetic pathway for this vitamin. The amoebae were starving on a diet of CoQ6 yeasts which could be complemented by either the addition of longer CoQs or the genetic engineering of a CoQ9 biosynthetic pathway.

Isolation of Naegleria lustrarea n. sp. (Excavata, Discoba, Heterolobosea) from the feces of Ambystoma annulatum (Ringed Salamander) in Northwest Arkansas.

The salamander, Ambystoma annulatum, is considered a "species of special concern" in the state of Arkansas, USA, due to its limited geographic range, specialized habitat requirements and low population size. Although metazoan parasites have been documented in this salamander species, neither its native protists nor microbiome have yet been evaluated. This is likely due to the elusive nature and under-sampling of the animal. Here, we initiate the cataloguing of microbial associates with the identification of a new heterlobosean species, Naegleria lustrarea n. sp. (Excavata, Discoba, Heterolobosea), isolated from feces of an adult A. annulatum.

High above the rest: standing behaviors in the amoebae of Sappinia and Thecamoeba.

Many terrestrial microbes have evolved cell behaviors that help them rise above their substrate, often to facilitate dispersal. One example of these behaviors is found in the amoebae of Sappinia pedata, which actively lift most of their cell mass above the substrate, known as standing. This standing behavior was first described in S. pedata in the 1890s from horse dung isolates but never molecularly characterized from dung. Our study expands this understanding, revealing the first molecularly confirmed S. pedata from herbivore dung in Mississippi, USA, and describing a new species, Sappinia dangeardi n. sp., with larger trophozoite cells. Additionally, we isolated another standing amoeba, Thecamoeba homeri n. sp., from soil, exhibiting a previously unreported "doughnut shape" transient behavior. In S. dangeardi n. sp., we discovered that standing is likely triggered by substrate drying, and that actin filaments actively localize in the "stalk" to support the standing cells, as observed through confocal microscopy. While the purpose of standing behaviors has not been investigated, we hypothesize it is energetically expensive and therefore a significant evolutionary strategy in these organisms. Overall, this study emphasizes behavioral adaptations to terrestrial environments within Amoebozoa, stressing the importance of diverse laboratory conditions that replicate natural habitats.

In the social amoeba Dictyostelium discoideum, shortened stalks may limit obligate cheater success even when exploitable partners are available.

Cooperation is widespread across life, but its existence can be threatened by exploitation. The rise of obligate social cheaters that are incapable of contributing to a necessary cooperative function can lead to the loss of that function. In the social amoeba Dictyostelium discoideum, obligate social cheaters cannot form dead stalk cells and in chimeras instead form living spore cells. This gives them a competitive advantage within chimeras. However, obligate cheaters of this kind have thus far not been found in nature, probably because they are often enough in clonal populations that they need to retain the ability to produce stalks. In this study we discovered an additional cost to obligate cheaters. Even when there are wild-type cells to parasitize, the chimeric fruiting bodies that result have shorter stalks and these are disadvantaged in spore dispersal. The inability of obligate cheaters to form fruiting bodies when they are on their own combined with the lower functionality of fruiting bodies when they are not represent limits on obligate social cheating as a strategy.

Neutrophils versus Protozoan Parasites: Plasmodium, Trichomonas, Leishmania, Trypanosoma, and Entameoba.

Neutrophils are the most abundant polymorphonuclear granular leukocytes in human blood and are an essential part of the innate immune system. Neutrophils are efficient cells that eliminate pathogenic bacteria and fungi, but their role in dealing with protozoan parasitic infections remains controversial. At sites of protozoan parasite infections, a large number of infiltrating neutrophils is observed, suggesting that neutrophils are important cells for controlling the infection. Yet, in most cases, there is also a strong inflammatory response that can provoke tissue damage. Diseases like malaria, trichomoniasis, leishmaniasis, Chagas disease, and amoebiasis affect millions of people globally. In this review, we summarize these protozoan diseases and describe the novel view on how neutrophils are involved in protection from these parasites. Also, we present recent evidence that neutrophils play a double role in these infections participating both in control of the parasite and in the pathogenesis of the disease.

Nitric oxide signaling through three receptors regulates virulence, biofilm formation, and phenotypic heterogeneity of Legionella pneumophila.

The causative agent of Legionnaires' disease, Legionella pneumophila, is an environmental bacterium, that replicates in macrophages, parasitizes amoeba, and forms biofilms. L. pneumophila employs the Legionella quorum sensing (Lqs) system and the transcription factor LvbR to control various bacterial traits, including virulence and biofilm architecture. LvbR negatively regulates the nitric oxide (NO) receptor Hnox1, linking quorum sensing to NO signaling. Here, we assessed the response of L. pneumophila to NO and investigated bacterial receptors underlying this process. Chemical NO donors, such as dipropylenetriamine (DPTA) NONOate and sodium nitroprusside (SNP), delayed and reduced the expression of the promoters for flagellin (PflaA) and the 6S small regulatory RNA (P6SRNA). Marker-less L. pneumophila mutant strains lacking individual (Hnox1, Hnox2, or NosP) or all three NO receptors (triple knockout, TKO) grew like the parental strain in media. However, in the TKO strain, the reduction of PflaA expression by DPTA NONOate was less pronounced, suggesting that the NO receptors are implicated in NO signaling. In the ΔnosP mutant, the lvbR promoter was upregulated, indicating that NosP negatively regulates LvbR. The single and triple NO receptor mutant strains were impaired for growth in phagocytes, and phenotypic heterogeneity of non-growing/growing bacteria in amoebae was regulated by the NO receptors. The single NO receptor and TKO mutant strains showed altered biofilm architecture and lack of response of biofilms to NO. In summary, we provide evidence that L. pneumophila regulates virulence, intracellular phenotypic heterogeneity, and biofilm formation through NO and three functionally non-redundant NO receptors, Hnox1, Hnox2, and NosP. IMPORTANCE: The highly reactive diatomic gas molecule nitric oxide (NO) is produced by eukaryotes and bacteria to promote short-range and transient signaling within and between neighboring cells. Despite its importance as an inter-kingdom and intra-bacterial signaling molecule, the bacterial response and the underlying components of the signaling pathways are poorly characterized. The environmental bacterium Legionella pneumophila forms biofilms and replicates in protozoan and mammalian phagocytes. L. pneumophila harbors three putative NO receptors, one of which crosstalks with the Legionella quorum sensing (Lqs)-LvbR network to regulate various bacterial traits, including virulence and biofilm architecture. In this study, we used pharmacological, genetic, and cell biological approaches to assess the response of L. pneumophila to NO and to demonstrate that the putative NO receptors are implicated in NO detection, bacterial replication in phagocytes, intracellular phenotypic heterogeneity, and biofilm formation.

Influence of the Age of Free-Living Amoeba Cysts on Their Vertical Distribution in a Water Column.

Free-living amoebae (FLA) are widely distributed protozoa in both natural and artificial environments such as drinking water. In addition to the ability of all FLA to transport various pathogenic microorganisms, certain species, such as Acanthamoeba spp. or Balamuthia mandrillaris, have intrinsic pathogenic abilities and cause severe cerebral infections. Previous work has shown an enrichment of FLA cysts in biofilm developed in upper levels of Drinking Water Storage Towers (DWSTs), suggesting that differences in densities of FLA cysts may play a role in their unequal distribution in the water column. To evaluate this hypothesis, a model of a water column was created for this study and used to analyze the vertical distribution of cysts of the FLA Acanthamoeba castellanii, Vermamoeba vermiformis, and Balamuthia mandrillaris from 0 to 23 weeks. Interestingly, our data showed that the cysts of both A. castellanii and V. vermiformis were enriched in upper water levels during their aging. However, B. mandrillaris cysts were equally distributed in the water column during the entire study. These results show that, in addition to the role of water level variation in the DWST, some FLA cysts can become less dense during their aging, which contributes to their enrichment in upper water and therefore biofilm levels.

Unusual and Unconsidered Mechanisms of Bacterial Resilience and Resistance to Quinolones.

Quinolone resistance has been largely related to the presence of specific point mutations in chromosomal targets, with an accessory role of impaired uptake and enhanced pump-out. Meanwhile the relevance of transferable mechanisms of resistance able to protect the target of pump-out or inactivate quinolones has been increasingly reported since 1998. Nevertheless, bacteria have other strategies and mechanisms allowing them to survive and even proliferate in the presence of quinolones, which might be qualified as resistance or resilience mechanisms. These include decreasing levels of quinolone target production, transient amoeba protection, benthonic lifestyle, nutrient-independent slow growth, activation of stringent response, inactivation or degradation of quinolones as well as apparently unrelated or forgotten chromosomal mutations. These mechanisms have been largely overlooked, either because of the use of classical approaches to antibiotic resistance determination or due to the low increase in final minimum inhibitory concentration levels. This article is devoted to a review of a series of these mechanisms.

Adaptation to an amoeba host drives selection of virulence-associated traits and genetic variation in saprotrophic Candida albicans.

Amoebae are micropredators that play an important role in controlling fungal populations in ecosystems. However, the interaction between fungi and their amoebic predators suggests that the pressure from predatory selection can significantly influence the development of fungal virulence and evolutionary processes. Thus, the purpose of this study was to investigate the adaptation of saprotrophic Candida albicans strains during their interactions with Acanthamoeba castellanii. We conducted a comprehensive analysis of survival after co-culture by colony counting of the yeast cells and examining yeast cell phenotypic and genetic characteristics. Our results indicated that exposure to amoebae enhanced the survival capacity of environmental C. albicans and induced visible morphological alterations in C. albicans, particularly by an increase in filamentation. These observed phenotypic changes were closely related to concurrent genetic variations. Notably, mutations in genes encoding transcriptional repressors (TUP1 and SSN6), recognized for their negative regulation of filamentous growth, were exclusively identified in amoeba-passaged isolates, and absent in unexposed isolates. Furthermore, these adaptations increased the exposed isolates' fitness against various stressors, simultaneously enhancing virulence factors and demonstrating an increased ability to invade A549 lung human epithelial cells. These observations indicate that the sustained survival of C. albicans under ongoing amoebic predation involved a key role of mutation events in microevolution to modulate the ability of these isolates to change phenotype and increase their virulence factors, demonstrating an enhanced potential to survive in diverse environmental niches.

A case report of Balamuthia mandrillaris encephalitis.

Balamuthia amoebic encephalitis (BAE) is a rare and severe parasitic infection of the central nervous system. Its delayed diagnosis and treatment are often due to the lack of specific clinical manifestations and its poor prognosis. Reported mortality rates reach around 95%. The Balamuthia mandrillaris is also known as the "brain-eating amoeba." Recently, the use of metagenomic next-generation sequencing (mNGS) in clinical settings has led to an increase in BAE diagnoses. A case report detailing the use of mNGS to diagnose granulomatous encephalitis caused by the Baramsi amoeba has improved clinicians' understanding of this disease and helped reduce misdiagnoses and missed diagnoses.

Growth dynamic of biofilm-associated Naegleria fowleri in freshwater on various materials.

In industrial water systems, the occurrence of biofilm-associated pathogenic free-living amoebae (FLA) such as Naegleria fowleri is a potential hygienic problem, and factors associated with its occurrence remain poorly understood. This study aimed to evaluate the impact of four cooling circuit materials on the growth of N. fowleri in a freshwater biofilm formed at 42°C and under a hydrodynamic shear rate of 17 s-1 (laminar flow): polyvinyl chloride, stainless steel, brass, and titanium. Colonization of the freshwater biofilms by N. fowleri was found to be effective on polyvinyl chloride, stainless steel, and titanium. For these three materials, the ratio of (bacterial prey)/(amoeba) was found to control the growth of N. fowleri. All materials taken together, a maximum specific growth rate of 0.18 ± 0.07 h-1 was associated with a generation time of ~4 h. In contrast, no significant colonization of N. fowleri was found on brass. Therefore, the contribution of copper is strongly suspected.

Prokaryotic microbial ecology as an ecosurveillance tool for eukaryotic pathogen colonisation: Meiothermus and Naegleria fowleri.

Naegleria fowleri has been detected in drinking water distribution systems (DWDS) in Australia, Pakistan and the United States and is the causative agent of the highly fatal disease primary amoebic meningoencephalitis. Previous small scale field studies have shown that Meiothermus may be a potential biomarker for N. fowleri. However, correlations between predictive biomarkers in small sample sizes often breakdown when applied to larger more representative datasets. This study represents one of the largest and most rigorous temporal investigations of Naegleria fowleri colonisation in an operational DWDS in the world and measured the association of Meiothermus and N. fowleri over a significantly larger space and time in the DWDS. A total of 232 samples were collected from five sites over three-years (2016-2018), which contained 29 positive N. fowleri samples. Two specific operational taxonomic units assigned to M. chliarophilus and M. hypogaeus, were significantly associated with N. fowleri presence. Furthermore, inoculation experiments demonstrated that Meiothermus was required to support N. fowleri growth in field-collected biofilms. This validates Meiothermus as prospective biological tool to aid in the identification and surveillance of N. fowleri colonisable sites.

Metagenomics insights into microbiome and antibiotic resistance genes from free living amoeba in chlorinated wastewater effluents.

Free living amoeba (FLA) are among the organisms commonly found in wastewater and are well-established hosts for diverse microbial communities. Despite its clinical significance, there is little knowledge on the FLA microbiome and resistome, with previous studies relying mostly on conventional approaches. In this study we comprehensively analyzed the microbiome, antibiotic resistome and virulence factors (VFs) within FLA isolated from final treated effluents of two wastewater treatment plants (WWTPs) using shotgun metagenomics. Acanthamoeba has been identified as the most common FLA, followed by Entamoeba. The bacterial diversity showed no significant difference (p > 0.05) in FLA microbiomes obtained from the two WWTPs. At phylum level, the most dominant taxa were Proteobacteria, followed by Firmicutes and Actinobacteria. The most abundant genera identified were Enterobacter followed by Citrobacter, Paenibacillus, and Cupriavidus. The latter three genera are reported here for the first time in Acanthamoeba. In total, we identified 43 types of ARG conferring resistance to cephalosporins, phenicol, streptomycin, trimethoprim, quinolones, cephalosporins, tigecycline, rifamycin, and kanamycin. Similarly, a variety of VFs in FLA metagenomes were detected which included flagellar proteins, Type IV pili twitching motility proteins (pilH and rpoN), alginate biosynthesis genes AlgI, AlgG, AlgD and AlgW and Type VI secretion system proteins and general secretion pathway proteins (tssM, tssA, tssL, tssK, tssJ, fha, tssG, tssF, tssC and tssB, gspC, gspE, gspD, gspF, gspG, gspH, gspI, gspJ, gspK, and gspM). To the best of our knowledge, this is the first study of its kind to examine both the microbiomes and resistome in FLA, as well as their potential pathogenicity in treated effluents. Additionally, this study showed that FLA can host a variety of potentially pathogenic bacteria including Paenibacillus, and Cupriavidus that had not previously been reported, indicating that their relationship may play a role in the spread and persistence of antibiotic resistant bacteria (ARBs) and antibiotic resistance genes (ARGs) as well as the evolution of novel pathogens.