Surviving chlorinated waters: bleaching sensitivity and persistence of free-living amoebae.

Recent advancements in membrane technologies and disinfection methods have enhanced drinking water quality significantly. However, microorganisms, including free-living amoebae (FLA), persist and pose potential threats to humans. FLA are linked to severe neuro-ophthalmic infections and serve as hosts of pathogenic bacteria. This study examined FLA presence in chlorinated and ultrafiltration drinking water and evaluated chlorine's disinfectant. Of 115 water samples, 21 tested positive for Acanthamoeba sp., Allovahlkampfia sp., and Vermamoeba vermiformis, originating from chlorinated sources. FLA trophozoites withstand temperatures up to 37 °C, while the cysts tolerate heat shocks of 60-70 °C. Trophozoites are susceptible to 5 mg L-1 chlorine, but cysts remain viable at concentrations up to 10 mg L-1. FLAs' survival in chlorinated waters is attributed to high cyst tolerance and lower residual chlorine concentrations. These findings highlight the need for ultrafiltration or enhanced chlorination protocols to ensure safer drinking water.

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.

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.

Occurrence of Free-Living Amoebae in Non-Human Primate Gut.

The gut microbiome reflects health and predicts possible disease in hosts. A holistic view of this community is needed, focusing on identifying species and dissecting how species interact with their host and each other, regardless of whether their presence is beneficial, inconsequential, or detrimental. The distribution of gut-associated eukaryotes within and across non-human primates is likely driven by host behavior and ecology. To ascertain the existence of free-living amoebae (FLA) in the gut of wild and captive non-human primates, 101 stool samples were collected and submitted to culture-dependent microscopy examination and DNA sequencing. Free-living amoebae were detected in 45.4% (46/101) of fecal samples analyzed, and their morphological characteristics matched those of Acanthamoeba spp., Vermamoeba spp., heterolobosean amoeboflagellates and fan-shaped amoebae of the family Vannellidae. Sequence analysis of the PCR products revealed that the suspected amoebae are highly homologous (99% identity and 100% query coverage) with Acanthamoeba T4 genotype and Vermamoeba vermiformis amoebae. The results showed a great diversity of amoebae in the non-human primate's microbiome, which may pose a potential risk to the health of NHPs. To our knowledge, this is the first report of free-living amoebae in non-human primates that are naturally infected. However, it is unknown whether gut-borne amoebae exploit a viable ecological niche or are simply transient residents in the gut.

Global prevalence of potentially pathogenic free-living amoebae in sewage and sewage-related environments-systematic review with meta-analysis.

Free-living amoebae (FLA) include amphizoic microorganisms important in public health, widely isolated from air, water, and soil. However, its occurrence in sewage-related environments still needs to be systematically documented. This study summarizes the occurrence of FLA in sewage-related environments through a systematic review with meta-analysis. A total of 1983 scientific article were retrieved from different databases, of which 35 were selected and analyzed using a random effects forest plot model with a 95% confidence interval (IC). The pooled overall prevalence of FLA in sewage across 12 countries was 68.96% (95% IC = 58.5-79.42). Subgroup analysis indicates high prevalence in all environments analyzed, including sewage water from the sewage treatment plant (81.19%), treated sewage water (75.57%), sewage-contaminated water (67.70%), sediment contaminated by sewage (48.91%), and sewage water (47.84%). Prevalence values of Acanthamoeba spp., Hartmanella/Vermamoeba spp., and Naegleria spp. are 47.48%, 28.24%, and 16.69%, respectively. Analyzing the species level, the distribution is as follows: Acanthamoeba palestinensis (88%), A. castellanii (23.74%), A. astronyxis (19.18%), A. polyphaga (13.59%), A. culbertsoni (12.5%), A. stevensoni (8.33%), A. tubiashi (4.35%) and A. hatchetti (1.1%), Naegleria fowleri (28.4%), N. gruberi (25%), N. clarki (8.33%), N. australiensis (4.89%) and N. italica (4.29%), Hartmannella/Vermamoeba exundans (40%) and H.V. vermiform (32.61%). Overall, our findings indicate a high risk associated with sewage-related environments, as the prevalence of FLA, including pathogenic strains, is high, even in treated sewage water. The findings of this study may be valuable both for risk remediation actions against amoebic infections and for future research endeavors.

Grazing preferences of three species of amoebae on cyanobacteria and green algae.

Twenty species/isolates of cyanobacteria and green algae were isolated from cyanobacterial bloom samples in lakes associated with the upper Qu'Appelle River drainage system in southern Saskatchewan, Canada. Three amoebae species (Cochliopodium sp., Vannella sp. and Vermamoeba vermiformis) were also isolated from one of these samples, and were subjected to grazing assays to determine which species of cyanobacteria or algae could potentially serve as a food source. Amoeba grazing rates were quantified based on the diameter of the plaque after 12 days on agar plate assays, and by estimation of the amoeba population growth rate from the rate of increase of plaque area. The common cyanobacterial bloom-formers Dolichospermum sp. and Aphanizomenon flos-aquae supported high growth rates for all three amoebae, while green algae, with the exception of one green alga/amoeba combination, did not support growth of the tested amoebae. Many of the cyanobacterial and algal isolates that did not support amoebae growth were ingested, suggesting that ingestion did not determine grazing success. Overall, while the cyanobacteria Dolichospermum sp. and Aphanizomenon flos-aquae were suitable food sources for the amoebae, the other cyanobacteria were grazed in an unpredictable manner, with some species/strains grazed by some amoebae and some species not grazed at all.

Global prevalence of free-living amoebae in solid matrices - A systematic review with meta-analysis.

The ubiquitous free-living amoebae (FLA) are microorganisms of significant medical, sanitary, and ecological importance. However, their characterization within solid matrices such as soil, dust, sediment, mud, sludge, and compost remain to be systematized. In this study, we conducted a systematic review with meta-analysis to explore the global distribution of FLA in solid matrices. From the analysis of 104 out of 4,414 scientific articles retrieved from different databases, it was found that the general global prevalence of FLA in solid matrices was of 55.13% (95% confidence interval (CI) 49.32-60.94). Specifically, FLA prevalence was high in soil (72.40%, 95% CI 69.08-75.73), sediment (57.91%, 95% CI 50.01-65.81), mud (52.90%, 95% CI 24.01-81.78), dust (48.60%, 95% CI 43.00-54.19), and sewage sludge (40.19%, 95% CI 30.68-49.70). In aerosols it was comparatively lower (17.21%, 95% CI 12.76-21.66). Acanthamoeba spp. (52.23%) and Hartmanella/Vermamoeba spp. (36.06%) were found to be more prevalent, whereas Naegleria spp. (34.98%) and Balamuthia spp. (27.32%) were less prevalent. The distribution of the highest global prevalence values for species of Acanthamoeba spp., considering different publication periods of the studies, is as follows: A. hatchetti (51.46%), A. rhysodes (47.49%), A. polyphaga (36.37%), A. culbertsoni (34.31%), A. castellanii (34.21%), and A. lenticulata (32.82%). For other FLA species, the distribution is: Hartmannella/Vermamoeba vermiformis (91.57%), Naegleria fowleri (42.32%), Naegleria gruberi (32.39%), and Balamuthia mandrillaris (25%). The most prevalent Acanthamoeba genotypes were T4 (33.38%) and T3 (23.94%). Overall, the global prevalence of FLA in solid matrices is as high as or greater than that reported in water by previous systematic reviews. Thus, actions aimed at reducing exposure to FLA or exploring their ecological dynamics should consider not only water but also the various solid matrices. The finding outlined here can provide valuable insights for such actions, e.g., informing on the level of exposure to FLA, or on the microbial biodiversity of specific environmental compartments.

Legionella pneumophila Rhizoferrin Promotes Bacterial Biofilm Formation and Growth within Amoebae and Macrophages.

Previously, we showed that Legionella pneumophila secretes rhizoferrin, a polycarboxylate siderophore that promotes bacterial growth in iron-deplete media and the murine lung. Yet, past studies failed to identify a role for the rhizoferrin biosynthetic gene (lbtA) in L. pneumophila infection of host cells, suggesting the siderophore's importance was solely linked to extracellular survival. To test the possibility that rhizoferrin's relevance to intracellular infection was missed due to functional redundancy with the ferrous iron transport (FeoB) pathway, we characterized a new mutant lacking both lbtA and feoB. This mutant was highly impaired for growth on bacteriological media that were only modestly depleted of iron, confirming that rhizoferrin-mediated ferric iron uptake and FeoB-mediated ferrous iron uptake are critical for iron acquisition. The lbtA feoB mutant, but not its lbtA-containing complement, was also highly defective for biofilm formation on plastic surfaces, demonstrating a new role for the L. pneumophila siderophore in extracellular survival. Finally, the lbtA feoB mutant, but not its complement containing lbtA, proved to be greatly impaired for growth in Acanthamoeba castellanii, Vermamoeba vermiformis, and human U937 cell macrophages, revealing that rhizoferrin does promote intracellular infection by L. pneumophila. Moreover, the application of purified rhizoferrin triggered cytokine production from the U937 cells. Rhizoferrin-associated genes were fully conserved across the many sequenced strains of L. pneumophila examined but were variably present among strains from the other species of Legionella. Outside of Legionella, the closest match to the L. pneumophila rhizoferrin genes was in Aquicella siphonis, another facultative intracellular parasite of amoebae.

Distribution and Current State of Molecular Genetic Characterization in Pathogenic Free-Living Amoebae.

Free-living amoebae (FLA) are protozoa widely distributed in the environment, found in a great diversity of terrestrial biomes. Some genera of FLA are linked to human infections. The genus Acanthamoeba is currently classified into 23 genotypes (T1-T23), and of these some (T1, T2, T4, T5, T10, T12, and T18) are known to be capable of causing granulomatous amoebic encephalitis (GAE) mainly in immunocompromised patients while other genotypes (T2, T3, T4, T5, T6, T10, T11, T12, and T15) cause Acanthamoeba keratitis mainly in otherwise healthy patients. Meanwhile, Naegleria fowleri is the causative agent of an acute infection called primary amoebic meningoencephalitis (PAM), while Balamuthia mandrillaris, like some Acanthamoeba genotypes, causes GAE, differing from the latter in the description of numerous cases in patients immunocompetent. Finally, other FLA related to the pathologies mentioned above have been reported; Sappinia sp. is responsible for one case of amoebic encephalitis; Vermamoeba vermiformis has been found in cases of ocular damage, and its extraordinary capacity as endocytobiont for microorganisms of public health importance such as Legionella pneumophila, Bacillus anthracis, and Pseudomonas aeruginosa, among others. This review addressed issues related to epidemiology, updating their geographic distribution and cases reported in recent years for pathogenic FLA.

Free-Living Amoeba Vermamoeba vermiformis Induces Allergic Airway Inflammation.

The high percentage of Vermamoeba was found in tap water in Korea. This study investigated whether Vermamoeba induced allergic airway inflammation in mice. We selected 2 free-living amoebas (FLAs) isolated from tap water, which included Korean FLA 5 (KFA5; Vermamoeba vermiformis) and 21 (an homolog of Acanthamoeba lugdunensis KA/ E2). We axenically cultured KFA5 and KFA21. We applied approximately 1 × 106 to mice's nasal passages 6 times and investigated their pathogenicity. The airway resistance value was significantly increased after KFA5 and KFA21 treatments. The eosinophil recruitment and goblet cell hyperplasia were concomitantly observed in bronchial alveolar lavage (BAL) fluid and lung tissue in mice infected with KFA5 and KFA21. These infections also activated the Th2-related interleukin 25, thymic stromal lymphopoietin, and thymus and activation-regulated chemokines gene expression in mouse lung epithelial cells. The CD4+ interleukin 4+ cell population was increased in the lung, and the secretion of Th2-, Th17-, and Th1-associated cytokines were upregulated during KFA5 and KFA21 infection in the spleen, lung-draining lymph nodes, and BAL fluid. The pathogenicity (allergenicity) of KFA5 and KFA21 might not have drastically changed during the long-term in vitro culture. Our results suggested that Vermamoeba could elicit allergic airway inflammation and may be an airway allergen.

Pathogenic free-living amoebae from water sources in Cape Verde.

Free-living amoebae (FLA) are protozoa which have been reported in different countries worldwide from diverse sources (water, soil, dust, air), contributing to the environmental microbiological contamination. Most of the FLA species present a life cycle with two different phases: an active vegetative and physiologically form named trophozoite, and an extremely resistant phase called cyst. Acanthamoeba spp., Naegleria fowleri, Balamuthia mandrillaris, Sapinia pedata, Vahlkampfia spp., Paravahlkampfia spp. and Vermamoeba vermiformis have been reported not only as causal agents of several opportunistic diseases including fatal encephalitis or epithelial disorders, but also as capable to favour the intracellular survival of common pathogenic bacteria, which could avoid the typical water disinfection systems, non-effective against FLAs cysts. Even though Santiago Island possesses high levels of humidity compared to the rest of the archipelago of Cape Verde, the water resources are scarce. Therefore, it is important to carry out proper microbiological quality controls, which currently do not contemplate the FLA presence in most of the countries. In the present work, we have reported the presence of Acanthamoeba spp. (69.2%); Vannella spp. (15.4%); Vermamoeba vermiformis (7.7%) and the recently discovered Stenamoeba dejonckheerei (7.7%) in different water sources of Santiago Island.

Diversity of Giant Viruses Infecting Vermamoeba vermiformis.

The discovery of Acanthamoeba polyphaga mimivirus in 2003 using the free-living amoeba Acanthamoeba polyphaga caused a paradigm shift in the virology field. Twelve years later, using another amoeba as a host, i.e., Vermamoeba vermiformis, novel isolates of giant viruses have been discovered. This amoeba-virus relationship led scientists to study the evolution of giant viruses and explore the origins of eukaryotes. The purpose of this article is to review all the giant viruses that have been isolated from Vermamoeba vermiformis, compare their genomic features, and report the influence of these viruses on the cell cycle of their amoebal host. To date, viruses putatively belonging to eight different viral taxa have been described: 7 are lytic and 1 is non-lytic. The comparison of giant viruses infecting Vermamoeba vermiformis has suggested three homogenous groups according to their size, the replication time inside the host cell, and the number of encoding tRNAs. This approach is an attempt at determining the evolutionary origins and trajectories of the virus; therefore, more giant viruses infecting Vermamoeba must be discovered and studied to create a comprehensive knowledge on these intriguing biological entities.

Compromised Effectiveness of Thermal Inactivation of Legionella pneumophila in Water Heater Sediments and Water, and Influence of the Presence of Vermamoeba vermiformis.

Intermittent reduction of temperature set-points and periodic shutdowns of water heaters have been proposed to reduce energy consumption in buildings. However, the consequences of such measures on the occurrence and proliferation of Legionella pneumophila (Lp) in hot water systems have not been documented. The impact of single and repeated heat shocks was investigated using an environmental strain of L. pneumophila and a reference strain of V. vermiformis. Heat shocks at temperatures ranging from 50 °C to 70 °C were applied for 1 h and 4 h in water and water heaters loose deposits (sludge). The regrowth potential of heat-treated culturable L. pneumophila in presence of V. vermiformis in water heaters sludges was evaluated. A 2.5-log loss of culturability of L. pneumophila was observed in simulated drinking water at 60 °C while a 4-log reduction was reached in water heaters loose deposits. Persistence of Lp after 4 h at 55 °C was shown and the presence of V. vermiformis in water heater's loose deposits resulted in a drastic amplification (5-log). Results show that thermal inactivation by heat shock is only efficient at elevated temperatures (50 °C) in both water and loose deposits. The few remaining organisms can rapidly proliferate during storage at lower temperature in the presence of hosts.

Diagnostic Considerations for Non-Acanthamoeba Amoebic Keratitis and Clinical Outcomes.

Cases of amoebic keratitis involving species other than Acanthamoeba are hypothesised to be underdiagnosed and poorly understood. Amoebic keratitis is debilitating and associated with chronic visual impairment. Understanding associated symptoms of non-Acanthamoeba amoebic keratitis could facilitate new diagnostic procedures and enable prompt treatment, ultimately leading to improved patient outcomes. Thus, a review of the literature was undertaken surrounding non-Acanthamoeba amoebic keratitis. Cases were geographically widespread and mostly confined to contact lens wearers ≤ 30 years old exposed to contaminated water sources and/or demonstrating poor lens hygiene. Vermamoeba vermiformis (previously Hartmanella vermiformis) was the most common causative agent, and a moderate number of mixed keratitis cases were also reported. A crucial disease indicator was early onset stromal deterioration/ulcerations, reported in 10 of the studies, usually only occurring in advanced Acanthamoeba keratitis. Mixed infections were the most difficult to treat, often requiring keratoplasty after unsuccessful combination treatment regimens. New diagnostic measures for non-Acanthamoeba amoebic keratitis should consider early onset stromal disease as a key disease indicator. Deep corneal scrapes are also necessary for accurate amoebic identification. Moreover, a combination approach to diagnosis is advised and should involve culture, microscopy and PCR techniques. In vitro drug sensitivity tests should also be conducted to help develop patient-specific treatment regimes.

Morphological and Genomic Features of the New Klosneuvirinae Isolate Fadolivirus IHUMI-VV54.

Since the discovery of Mimivirus, viruses with large genomes encoding components of the translation machinery and other cellular processes have been described as belonging to the nucleocytoplasmic large DNA viruses. Recently, genome-resolved metagenomics led to the discovery of more than 40 viruses that have been grouped together in a proposed viral subfamily named Klosneuvirinae. Members of this group had genomes of up to 2.4Mb in size and featured an expanded array of translation system genes. Yet, despite the large diversity of the Klosneuvirinae in metagenomic data, there are currently only two isolates available. Here, we report the isolation of a novel giant virus known as Fadolivirus from an Algerian sewage site and provide morphological data throughout its replication cycle in amoeba and a detailed genomic characterization. The Fadolivirus genome, which is more than 1.5Mb in size, encodes 1,452 predicted proteins and phylogenetic analyses place this viral isolate as a near relative of the metagenome assembled Klosneuvirus and Indivirus. The genome encodes for 66 tRNAs, 23 aminoacyl-tRNA synthetases and a wide range of transcription factors, surpassing Klosneuvirus and other giant viruses. The Fadolivirus genome also encodes putative vacuolar-type proton pumps with the domains D and A, potentially constituting a virus-derived system for energy generation. The successful isolation of Fadolivirus will enable future hypothesis-driven experimental studies providing deeper insights into the biology of the Klosneuvirinae.

The increasing importance of Vermamoeba vermiformis.

Vermamoeba vermiformis are one of the most prevalent free-living amoebae. These amoebae are ubiquitous and also thermotolerant. Of concern, V. vermiformis have been found in hospital water networks. Furthermore, associations between V. vermiformis and pathogenic bacteria have been reported, such as Legionella pneumophila. Moreover, V. vermiformis are well known to host viruses, bacteria, and other microorganisms and cases of keratitis due to V. vermiformis in conjunction with other amoebae have been reported. Despite the preceding, the medical importance of V. vermiformis is still an ongoing discussion and its genome has been only recently sequenced. Herein, we present a review of the current understanding of the biology and pathogenesis pertaining to V. vermiformis, as well as its' role as an etiological agent and trojan horse. An approach known as theranostics which combines both diagnosis and therapy could be utilized to eradicate and diagnose keratitis cases caused by such amoebae. Given the rise in global warming, it is imperative to investigate these rarely studied amoebae and to understand their importance in human health.

The Kaumoebavirus LCC10 Genome Reveals a Unique Gene Strand Bias among "Extended Asfarviridae".

Kaumoebavirus infects the amoeba Vermamoeba vermiformis and has recently been described as a distant relative of the African swine fever virus. To characterize the diversity and evolution of this novel viral genus, we report here on the isolation and genome sequencing of a second strain of Kaumoebavirus, namely LCC10. Detailed analysis of the sequencing data suggested that its 362-Kb genome is linear with covalently closed hairpin termini, so that DNA forms a single continuous polynucleotide chain. Comparative genomic analysis indicated that although the two sequenced Kaumoebavirus strains share extensive gene collinearity, 180 predicted genes were either gained or lost in only one genome. As already observed in another distant relative, i.e., Faustovirus, which infects the same host, the center and extremities of the Kaumoebavirus genome exhibited a higher rate of sequence divergence and the major capsid protein gene was colonized by type-I introns. A possible role of the Vermamoeba host in the genesis of these evolutionary traits is hypothesized. The Kaumoebavirus genome exhibited a significant gene strand bias over the two-third of genome length, a feature not seen in the other members of the "extended Asfarviridae" clade. We suggest that this gene strand bias was induced by a putative single origin of DNA replication located near the genome extremity that imparted a selective force favoring the genes positioned on the leading strand.

Busting biofilms: free-living amoebae disrupt preformed methicillin-resistant Staphylococcus aureus (MRSA) and Mycobacterium bovis biofilms.

Biofilm-associated infections are difficult to eradicate because of their ability to tolerate antibiotics and evade host immune responses. Amoebae and/or their secreted products may provide alternative strategies to inhibit and disperse biofilms on biotic and abiotic surfaces. We evaluated the potential of five predatory amoebae - Acanthamoeba castellanii, Acanthamoeba lenticulata, Acanthamoeba polyphaga, Vermamoeba vermiformis and Dictyostelium discoideum - and their cell-free secretions to disrupt biofilms formed by methicillin-resistant Staphylococcus aureus (MRSA) and Mycobacterium bovis. The biofilm biomass produced by MRSA and M. bovis was significantly reduced when co-incubated with A. castellanii, A. lenticulata and A. polyphaga, and their corresponding cell-free supernatants (CFS). Acanthamoeba spp. generally produced CFS that mediated biofilm dispersal rather than directly killing the bacteria; however, A. polyphaga CFS demonstrated active killing of MRSA planktonic cells when the bacteria were present at low concentrations. The active component(s) of the A. polyphaga CFS is resistant to freezing, but can be inactivated to differing degrees by mechanical disruption and exposure to heat. D. discoideum and its CFS also reduced preformed M. bovis biofilms, whereas V. vermiformis only decreased M. bovis biofilm biomass when amoebae were added. These results highlight the potential of using select amoebae species or their CFS to disrupt preformed bacterial biofilms.

Identification of Free-Living Amoebas in Tap Water of Buildings with Storage Tanks in Korea.

Free-living amoebas (FLAs) can cause severe disease in humans and animals when they become infected. However, there are no accurate survey reports on the prevalence of FLAs in Korea. In this study, we collected 163 tap water samples from buildings, apartments, and restrooms of highway service areas in 7 Korean provinces with high population density. All these buildings and facilities have water storage tanks in common. The survey was separated into categories of buildings, apartments, and highway service areas. Five hundred milliliters of tap water from each building was collected and filtered with 0.2 µm pore filter paper. The filters were incubated in agar plates with heated E. coli at 25°C. After axenization, genomic DNA was collected from each FLA, and species classification was performed using partial 18S-rDNA PCR-sequencing analysis. We found that 12.9% of tap water from buildings with storage tanks in Korea was contaminated with FLAs. The highway service areas had the highest contamination rate at 33.3%. All of the FLAs, except one, were genetically similar to Vermamoeba vermiformis (Hartmannella vermiformis). The remaining FLA (KFA21) was very similar to Acanthamoeba lugdunensis (KA/E26). Although cases of human infection by V. vermiformis are very rare, we must pay attention to the fact that one-third of tap water supplies in highway service areas have been contaminated.

Legionella pneumophila and Protozoan Hosts: Implications for the Control of Hospital and Potable Water Systems.

Legionella pneumophila is an opportunistic waterborne pathogen of public health concern. It is the causative agent of Legionnaires' disease (LD) and Pontiac fever and is ubiquitous in manufactured water systems, where protozoan hosts and complex microbial communities provide protection from disinfection procedures. This review collates the literature describing interactions between L. pneumophila and protozoan hosts in hospital and municipal potable water distribution systems. The effectiveness of currently available water disinfection protocols to control L. pneumophila and its protozoan hosts is explored. The studies identified in this systematic literature review demonstrated the failure of common disinfection procedures to achieve long term elimination of L. pneumophila and protozoan hosts from potable water. It has been demonstrated that protozoan hosts facilitate the intracellular replication and packaging of viable L. pneumophila in infectious vesicles; whereas, cyst-forming protozoans provide protection from prolonged environmental stress. Disinfection procedures and protozoan hosts also facilitate biogenesis of viable but non-culturable (VBNC) L. pneumophila which have been shown to be highly resistant to many water disinfection protocols. In conclusion, a better understanding of L. pneumophila-protozoan interactions and the structure of complex microbial biofilms is required for the improved management of L. pneumophila and the prevention of LD.