Genome Dynamics and Temperature Adaptation During Experimental Evolution of Obligate Intracellular Bacteria.

Evolution experiments with free-living microbes have radically improved our understanding of genome evolution and how microorganisms adapt. Yet there is a paucity of such research focusing on strictly host-associated bacteria, even though they are widespread in nature. Here, we used the Acanthamoeba symbiont Protochlamydia amoebophila, a distant relative of the human pathogen Chlamydia trachomatis and representative of a large group of protist-associated environmental chlamydiae, as a model to study how obligate intracellular symbionts evolve and adapt to elevated temperature, a prerequisite for the pivotal evolutionary leap from protist to endothermic animal hosts. We established 12 replicate populations under two temperatures (20 °C, 30 °C) for 510 bacterial generations (38 months). We then used infectivity assays and pooled whole-genome resequencing to identify any evolved phenotypes and the molecular basis of adaptation in these bacteria. We observed an overall reduction in infectivity of the symbionts evolved at 30 °C, and we identified numerous nonsynonymous mutations and small indels in these symbiont populations, with several variants persisting throughout multiple time points and reaching high frequencies. This suggests that many mutations may have been beneficial and played an adaptive role. Mutated genes within the same temperature regime were more similar than those between temperature regimes. Our results provide insights into the molecular evolution of intracellular bacteria under the constraints of strict host dependance and highly structured populations and suggest that for chlamydial symbionts of protists, temperature adaptation was facilitated through attenuation of symbiont infectivity as a tradeoff to reduce host cell burden.

Stagnation arising through intermittent usage is associated with increased viable but non culturable Legionella and amoeba hosts in a hospital water system.

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.

Acanthamoeba, an environmental phagocyte enhancing survival and transmission of human pathogens.

The opportunistic protist Acanthamoeba, which interacts with other microbes such as bacteria, fungi, and viruses, shows significant similarity in cellular and functional aspects to human macrophages. Intracellular survival of microbes in this microbivorous amoebal host may be a crucial step for initiation of infection in higher eukaryotic cells. Therefore, Acanthamoeba-microbe adaptations are considered an evolutionary model of macrophage-pathogen interactions. This paper reviews Acanthamoeba as an emerging human pathogen and different ecological interactions between Acanthamoeba and microbes that may serve as environmental training grounds and a genetic melting pot for the evolution, persistence, and transmission of potential human pathogens.

Use of a Novel DNA-Loaded Alginate-Calcium Carbonate Biopolymer Surrogate to Study the Engulfment of Legionella pneumophila by Acanthamoeba polyphaga in Water Systems.

The engulfment of Legionella pneumophila by free-living amoebae (FLA) in engineered water systems (EWS) enhances L. pneumophila persistence and provides a vehicle for rapid replication and increased public health risk. Despite numerous legionellosis outbreaks worldwide, effective tools for studying interactions between L. pneumophila and FLA in EWS are lacking. To address this, we have developed a biopolymer surrogate with a similar size, shape, surface charge, and hydrophobicity to those of stationary-phase L. pneumophila. Parallel experiments were conducted to observe the engulfment of L. pneumophila and the surrogate by Acanthamoeba polyphaga in dechlorinated, filter-sterilised tap water at 30°C for 72 h. Trophozoites engulfed both the surrogate and L. pneumophila, reaching maximum uptake after 2 and 6 h, respectively, but the peak surrogate uptake was ~2-log lower. Expulsion of the engulfed surrogate from A. polyphaga was also faster compared to that of L. pneumophila. Confocal laser scanning microscopy confirmed that the surrogate was actively engulfed and maintained within vacuoles for several hours before being expelled. L. pneumophila and surrogate phagocytosis appear to follow similar pathways, suggesting that the surrogate can be developed as a useful tool for studying interactions between L. pneumophila and FLA in EWS. IMPORTANCE The internalization of L. pneumophila within amoebae is a critical component of their life cycle in EWS, as it protects the bacteria from commonly used water disinfectants and provides a niche for their replication. Intracellularly replicated forms of L. pneumophila are also more virulent and resistant to sanitizers. Most importantly, the bacteria's adaptation to the intracellular environments of amoebae primes them for the infection of human macrophages, posing a significant public health risk in EWS. The significance of our study is that a newly developed L. pneumophila biopolymer surrogate can mimic the L. pneumophila engulfment process in A. polyphaga, a free-living amoeba. With further development, the surrogate has the potential to improve the understanding of amoeba-mediated L. pneumophila persistence in EWS and the associated public health risk management.

A Comparative Genomic Approach to Determine the Virulence Factors and Horizontal Gene Transfer Events of Clinical Acanthamoeba Isolates.

Acanthamoeba species are among the most ubiquitous protists that are widespread in soil and water and act as both a replicative niche and vectors for dispersal. They are the most important human intracellular pathogens, causing Acanthamoeba keratitis (AK) and severely damaging the human cornea. The sympatric lifestyle within the host and amoeba-resisting microorganisms (ARMs) promotes horizontal gene transfer (HGT). However, the genomic diversity of only A. castellanii and A. polyphaga has been widely studied, and the pathogenic mechanisms remain unknown. Thus, we examined 7 clinically pathogenic strains by comparative genomic, phylogenetic, and rhizome gene mosaicism analyses to explore amoeba-symbiont interactions that possibly contribute to pathogenesis. Genetic characterization and phylogenetic analysis showed differences in functional characteristics between the "open" state of T3 and T4 isolates, which may contribute to the differences in virulence and pathogenicity. Through comparative genomic analysis, we identified potential genes related to virulence, such as metalloprotease, laminin-binding protein, and HSP, that were specific to the genus Acanthamoeba. Then, analysis of putative sequence trafficking between Acanthamoeba and Pandoraviruses or Acanthamoeba castellanii medusaviruses provided the best hits with viral genes; among bacteria, Pseudomonas had the most significant numbers. The most parsimonious evolutionary scenarios were between Acanthamoeba and endosymbionts; nevertheless, in most cases, the scenarios are more complex. In addition, the differences in exchanged genes were limited to the same family. In brief, this study provided extensive data to suggest the existence of HGT between Acanthamoeba and ARMs, explaining the occurrence of diseases and challenging Darwin's concept of eukaryotic evolution. IMPORTANCEAcanthamoeba has the ability to cause serious blinding keratitis. Although the prevalence of this phenomenon has increased in recent years, our knowledge of the underlying opportunistic pathogenic mechanism maybe remains incomplete. In this study, we highlighted the importance of Pseudomonas in the pathogenesis pathway using comprehensive a whole genomics approach of clinical isolates. The horizontal gene transfer events help to explain how endosymbionts contribute Acanthamoeba to act as an opportunistic pathogen. Our study opens up several potential avenues for future research on the differences in pathogenicity and interactions among clinical strains.

Comparative analysis of differentially expressed genes in Acanthamoeba after ingestion of Legionella pneumophila and Escherichia coli.

Acanthamoeba spp. feeds on bacteria, fungi, and algae to obtain nutrients from the environment. However, several pathogens can survive and multiply in Acanthamoeba. Mechanisms necessary for the survival and proliferation of microorganisms in Acanthamoeba remain unclear. The object of this study was to identify effective factors for the survival of microorganisms in Acanthamoeba. Differentially expressed genes (DEGs) in A. castellanii infected by Legionella pneumophila or Escherichia coli were identified based on mRNA sequencing. A total of 2342 and 1878 DEGs were identified in Acanthamoeba with L. pneumophila and E. coli, respectively. Among these DEGs, 502 were up-regulated and 116 were down-regulated in Acanthamoeba infected by L. pneumophila compared to those in Acanthamoeba feed on E. coli. Gene ontology analysis showed that the genes encoded small GTPase-mediated signal transduction proteins in the biological process domain, intracellular proteins in the cellular component domain, and ATP binding proteins in the molecular function domain were up-regulated while integral components of membrane proteins in the cellular component domain were down-regulated in Acanthamoeba infected by Legionella compared to those in Acanthamoeba feed on E. coli. During endosymbiosis with Legionella, Acanthamoeba showed various changes in the expression of genes supposed to be involved in phagosomal maturation. Acanthamoeba infected by Legionella also showed high expression levels of aminotransferase, methyltransferase, and cysteine proteinase but low expression levels of RNA pseudouridine synthase superfamily protein and 2OG-Fe(II) oxygenase superfamily. These results provide directions for further research to understand the survival strategy of L. pneumophila in A. castellanii.

Pangenomics reveals alternative environmental lifestyles among chlamydiae.

Chlamydiae are highly successful strictly intracellular bacteria associated with diverse eukaryotic hosts. Here we analyzed metagenome-assembled genomes of the "Genomes from Earth's Microbiomes" initiative from diverse environmental samples, which almost double the known phylogenetic diversity of the phylum and facilitate a highly resolved view at the chlamydial pangenome. Chlamydiae are defined by a relatively large core genome indicative of an intracellular lifestyle, and a highly dynamic accessory genome of environmental lineages. We observe chlamydial lineages that encode enzymes of the reductive tricarboxylic acid cycle and for light-driven ATP synthesis. We show a widespread potential for anaerobic energy generation through pyruvate fermentation or the arginine deiminase pathway, and we add lineages capable of molecular hydrogen production. Genome-informed analysis of environmental distribution revealed lineage-specific niches and a high abundance of chlamydiae in some habitats. Together, our data provide an extended perspective of the variability of chlamydial biology and the ecology of this phylum of intracellular microbes.

War of the microbial world: Acanthamoeba spp. interactions with microorganisms.

Acanthamoeba is known to interact with a plethora of microorganisms such as bacteria, fungi and viruses. In these interactions, the amoebae can be predatory in nature, transmission vehicle or an incubator. Amoebae consume microorganisms, especially bacteria, as food source to fulfil their nutritional needs by taking up bacteria through phagocytosis and lysing them in phagolysosomes and hence play an eminent role in the regulation of bacterial density in the nature and accountable for eradication of around 60% of the bacterial population in the environment. Acanthamoeba can also act as a "Trojan horse" for microbial transmission in the environment. Additionally, Acanthamoeba may serve as an incubator-like reservoir for microorganisms, including those that are pathogenic to humans, where the microorganisms use amoebae's defences to resist harsh environment and evade host defences and drugs, whilst growing in numbers inside the amoebae. Furthermore, amoebae can also be used as a "genetic melting pot" where exchange of genes as well as adaptation of microorganisms, leading to higher pathogenicity, may arise. Here, we describe bacteria, fungi and viruses that are known to interact with Acanthamoeba spp.

Combining Whole-Genome Sequencing and Multimodel Phenotyping To Identify Genetic Predictors of Salmonella Virulence.

Salmonella comprises more than 2,600 serovars. Very few environmental and uncommon serovars have been characterized for their potential role in virulence and human infections. A complementary in vitro and in vivo systematic high-throughput analysis of virulence was used to elucidate the association between genetic and phenotypic variations across Salmonella isolates. The goal was to develop a strategy for the classification of isolates as a benchmark and predict virulence levels of isolates. Thirty-five phylogenetically distant strains of unknown virulence were selected from the Salmonella Foodborne Syst-OMICS (SalFoS) collection, representing 34 different serovars isolated from various sources. Isolates were evaluated for virulence in 4 complementary models of infection to compare virulence traits with the genomics data, including interactions with human intestinal epithelial cells, human macrophages, and amoeba. In vivo testing was conducted using the mouse model of Salmonella systemic infection. Significant correlations were identified between the different models. We identified a collection of novel hypothetical and conserved proteins associated with isolates that generate a high burden. We also showed that blind prediction of virulence of 33 additional strains based on the pan-genome was high in the mouse model of systemic infection (82% agreement) and in the human epithelial cell model (74% agreement). These complementary approaches enabled us to define virulence potential in different isolates and present a novel strategy for risk assessment of specific strains and for better monitoring and source tracking during outbreaks.IMPORTANCESalmonella species are bacteria that are a major source of foodborne disease through contamination of a diversity of foods, including meat, eggs, fruits, nuts, and vegetables. More than 2,600 different Salmonella enterica serovars have been identified, and only a few of them are associated with illness in humans. Despite the fact that they are genetically closely related, there is enormous variation in the virulence of different isolates of Salmonella enterica Identification of foodborne pathogens is a lengthy process based on microbiological, biochemical, and immunological methods. Here, we worked toward new ways of integrating whole-genome sequencing (WGS) approaches into food safety practices. We used WGS to build associations between virulence and genetic diversity within 83 Salmonella isolates representing 77 different Salmonella serovars. Our work demonstrates the potential of combining a genomics approach and virulence tests to improve the diagnostics and assess risk of human illness associated with specific Salmonella isolates.

War on Terror Cells: Strategies to Eradicate "Novel Coronavirus" Effectively.

Brain-eating amoebae are known to harbor a plethora of viral, bacterial, protozoal, and fungal pathogens and safeguard these pathogens against disinfectants. Due to their ubiquitous distribution in the environment and their status as the trojan horse of the microbial world, amoebae can provide novel coronavirus a means to susceptible hosts and possible transmission to the central nervous system. Here, we hypothesize that pursuing the host that harbor "terror cells" is a valuable approach in eradicating novel coronavirus in affected communities.

Applying of bacteria Cellulosimicrobium sp. for cultivation protozoa of genus Acanthamoeba

The aim of this study was identification features of cultivation representatives of genus Acanthamoeba isolated from bentonite using Cellulosimicrobium sp. as a bacteria-feeders. Identification of isolated bacteria was conducted by morphological, cultural and molecular-genetic methods. The cultivation of free-living "bentonite" amoeba on the lawn of Cellulosimicrobium sp. have gained significant advantages than using Escherichia coli as a bacteriafeeders was shown. "Bentonite" amoeba form crateroid plaques, which fit to the quantitative characteristic materials which contains amoeba, during deep co-cultivation Acanthamoeba sp. and Cellulosimicrobium sp. on 1% glucose meet-peptone agar.

Detección de Acanthamoeba por reacción en cadena de la polimerasa en tiempo real en líquidos de conservación de lentes de contacto / Detection of Acanthamoeba sp. by real-time polymerase chain reaction in contact lens solutions

RESUMEN Objetivo: Estandarizar una técnica de reacción en cadena de la polimerasa en tiempo real para la detección del parásito e identificar Acanthamoeba en líquidos conservantes de lentes de contacto. Métodos: Se realizó un estudio descriptivo observacional de corte transversal sobre la técnica de reacción en cadena de la polimerasa en tiempo real para la detección de Acanthamoeba, en el Instituto de Investigaciones en Ciencias de la Salud de la ciudad de Asunción, en Paraguay. Se analizaron 110 líquidos conservantes aportados por usuarios sanos de lentes de contacto, mediante reacción en cadena de la polimerasa en tiempo real y cultivo en medio PAGE - SDS. Resultados: Se estandarizó con éxito la técnica de reacción en cadena de la polimerasa en tiempo real con límite de sensibilidad de 1 pg/µL. Se aisló Acanthamoeba a partir de una muestra (1 por ciento) por método de cultivo, mientras que la carga parasitaria en el líquido conservante fue inferior al límite de detección de la reacción en cadena de la polimerasa en tiempo real. El ADN obtenido del cultivo de dicha muestra fue positivo para Acanthamoeba por este método. Conclusión: El sistema estandarizado presenta buena sensibilidad y podrá ser incorporado en los laboratorios que cuentan con acceso a equipos de reacción en cadena de la polimerasa en tiempo real para un diagnóstico rápido y más eficiente en casos de sospechas de queratitis amebiana. Recomendamos el uso combinado de métodos moleculares y cultivo para aumentar la potencia del diagnóstico, sobre todo en muestras donde la carga parasitaria es muy baja(AU)

ABSTRACT Objective: Standardize a real-time polymerase chain reaction technique for detection of the parasite and identify Acanthamoeba in contact lens solutions. Methods: A cross-sectional observational descriptive study was conducted about a real-time polymerase chain reaction technique for detection of Acanthamoeba at the Institute of Health Sciences Research in the city of Asunción, Paraguay. A total 110 solutions were analyzed, which were provided by healthy contact lens users, by real-time polymerase chain reaction and culture in SDS-PAGE medium. Results: Successful standardization was achieved of the real-time polymerase chain reaction technique with a sensitivity limit of 1 pg/µl. Acanthamoeba was isolated from one sample (1 percent) by culture, whereas the parasite load in the contact lens solution was below the detection limit of the real-time polymerase chain reaction technique. The DNA obtained from the culture of that sample was positive for Acanthamoeba by the real-time polymerase chain reaction technique method. Conclusion: The system standardized exhibits good sensitivity and may be incorporated into laboratories with real-time polymerase chain reaction technique equipment for a rapid and more efficient diagnosis of suspected amoebic keratitis. We recommend the combined use of molecular methods and culture to enhance diagnostic power, mainly in samples where the parasite load is very low(AU)

Complete genome and bimodal genomic structure of the amoebal symbiont Neochlamydia strain S13 revealed by ultra-long reads obtained from MinION.

Neochlamydia strain S13 is an amoebal symbiont of an Acanthamoeba sp. The symbiont confers resistance to Legionella pneumophila on its host; however, the molecular mechanism underlying this resistance is not completely understood. Genome analyses have been crucial for understanding the complicated host-symbiont relationship but segregating the host's genome DNA from the symbiont's DNA is often challenging. In this study, we successfully identified a bimodal genomic structure in Neochlamydia strain S13 using PacBio RS II supported by ultra-long reads derived from MinION. One mode consisted of circular sequences of 2,586,667 and 231,307 bp; the other was an integrated sequence of the two via long homologous regions. They encoded 2175 protein-coding regions, some of which were implied to be acquired via horizontal gene transfer. They were specifically conserved in the genus Neochlamydia and formed a cluster in the genome, presumably by multiplication through genome replication. Moreover, it was notable that the sequenced DNA was obtained without segregating the symbiont DNA from the host. This is an easy and versatile technique that facilitates the characterization of diverse hosts and symbionts in nature.

Interactions of free-living amoebae with the rice fungal pathogen, Rhizoctonia solani.

OBJECTIVE: Rhizoctonia solani is a soil-borne fungal pathogen of many important crop plants. In rice, R. solani causes sheath blight disease, which results in devastating grain yield and quality losses. Few methods are available to control this pathogen and classic single gene resistance mechanisms in rice plants have not been identified. We hypothesize that alternate means of control are available in the environment including free-living amoebae. Amoebae are soil-, water- and air-borne microorganisms that are predominantly heterotrophic. Many amoeba species are mycophagous, and several harm their prey using mechanisms other than phagocytosis. Here, we used light and scanning electron microscopy to survey the interactions of R. solani with four amoeba species, with the goal of identifying amoebae species with potential for biocontrol. RESULTS: We observed a wide range of responses during interactions of R. solani with four different free-living amoebae. Two Acanthamoeba species encyst in co-cultures with R. solani at higher rates than medium without R. solani. Vermamoeba vermiformis (formerly Hartmanella vermiformis) attach to R. solani mycelium and are associated with mycelial shriveling and perforations of fungal cell walls, indicating an antagonistic interaction. No phenotypic changes were observed in co-cultures of Dictyostelium discoideum and R. solani.

Screen for fitness and virulence factors of Francisella sp. strain W12-1067 using amoebae.

Francisella tularensis is the causative agent of the human disease referred to as tularemia. Other Francisella species are known but less is understood about their virulence factors. The role of environmental amoebae in the life-cycle of Francisella is still under discussion. Francisella sp. strain W12-1067 (F-W12) is an environmental Francisella isolate recently identified in Germany which is negative for the Francisella pathogenicity island, but exhibits a putative alternative type VI secretion system. Putative virulence factors have been identified in silico in the genome of F-W12. In this work, we established a "scatter screen", used earlier for pathogenic Legionella, to verify experimentally and identify candidate fitness factors using a transposon mutant bank of F-W12 and Acanthamoeba lenticulata as host organism. In these experiments, we identified 79 scatter clones (amoeba sensitive), which were further analyzed by an infection assay identifying 9 known virulence factors, but also candidate fitness factors of F-W12 not yet described as fitness factors in Francisella. The majority of the identified genes encoded proteins involved in the synthesis or maintenance of the cell envelope (LPS, outer membrane, capsule) or in the metabolism (glycolysis, gluconeogenesis, pentose phosphate pathway). Further 13C-flux analysis of the Tn5 glucokinase mutant strain revealed that the identified gene indeed encodes the sole active glucokinase in F-W12. In conclusion, candidate fitness factors of the new Francisella species F-W12 were identified using the scatter screen method which might also be usable for other Francisella species.

Interactions of Pseudomonas aeruginosa with Acanthamoeba polyphaga Observed by Imaging Flow Cytometry.

Pseudomonas aeruginosa is a Gram-negative bacterium that is abundant in the environment and water systems, with strains that cause serious infections, especially in patients with compromised immune systems. In times of stress or as part of its natural life cycle, P. aeruginosa can adopt a viable but not culturable (VBNC) state, which renders it undetectable by current conventional food and water testing methods and makes it highly resistant to antibiotic treatment. Specific conditions can resuscitate these coccoid VBNC P. aeruginosa cells, which returns them to their active, virulent rod-shaped form. Underreporting the VBNC cells of P. aeruginosa by standard culture-based methods in water distribution systems may therefore pose serious risks to public health. As such, being able to accurately detect and quantify the presence of VBNC P. aeruginosa, especially in a hospital setting, is of critical importance. Herein, we describe a method to analyze VBNC P. aeruginosa using imaging flow cytometry. With this technique, we can accurately distinguish between active and VBNC forms. We also show here that association of VBNC P. aeruginosa with Acanthamoeba polyphaga results in resuscitation of P. aeruginosa to an active form within 2 h. Our approach could provide an alternative, reliable detection method of VBNC P. aeruginosa when coupled with species-specific staining. Most importantly, our experiments demonstrate that the coculture with amoebae can lead to a resuscitation of P. aeruginosa of culturable morphology after only 2 h, indicating that VBNC P. aeruginosa could potentially resuscitate in piped water (healthcare) environments colonized with amoebae. © 2019 International Society for Advancement of Cytometry.

Molecular characterization of bacterial, viral and fungal endosymbionts of Acanthamoeba isolates in keratitis patients of Iran.

Free-living amoebae belong to the genus Acanthamoeba; can feed on microbial population by phagocytosis, and with the capability to act as a reservoir and a vehicle of microorganisms to susceptible host. Therefore, the role of endosymbiosis in the pathogenesis of Acanthamoeba is complex and not fully understood. The aim of the present study was to identify bacterial, fungal, and human adenovirus (HADV) endosymbionts as well as evaluating the endosymbionts role of such organisms in the pathogenesis of Acanthamoeba in keratitis patients living in Iran. Fifteen Acanthamoeba (T4 genotype) isolates were recovered from corneal scrapes and contact lenses of patients with keratitis. Cloning and purification was performed for all isolate. Gram staining was performed to identify bacterial endosymbionts. DNA extraction, PCR, and nested PCR was set up to identify endosymbiont of amoeba. Evaluation of pathogenicity was conducted by osmo-tolerance and thermo-tolerance assays and cell culture, and then CPE (cytopathic effect) was survey. Statistical analysis was used between Acanthamoeba associated endosymbionts and Acanthamoeba without endosymbiont at 24, 48, 72, and 96 h. A p value < 0.05 was considered as significant, statistically. A total of 9 (60%) Acanthamoeba (T4 genotypes) isolates were successfully cloned for detecting microorganism endosymbionts. The only isolate negative for the presence of endosymbiont was ICS9. ICS7 (Pseudomonas aeruginosa, Aspergillus sp., and human adenovirus endosymbionts) and ICS2 (Escherichia coli endosymbiont) isolates were considered as Acanthamoeba associated endosymbionts. ICS7 and ICS2 isolates were highly pathogen whereas ICS9 isolate showed low pathogenicity in pathogenicity evaluated. Positive CPE for ICS7 and ICS2 isolates and negative CPE for ICS9 isolate were observed in cell culture. The average number of cells, trophozoites, and cysts among ICS7, ICS2, and ICS9 isolates at 24, 48, 72, and 96 h was significant. This is the first survey on microbial endosymbionts of Acanthamoeba in keratitis patients of Iran, and also the first report of Aspergillus sp, Achromobacter sp., Microbacterium sp., Brevibacillus sp, Brevundimonas sp and Mastadenovirus sp in Acanthamoeba as endosymbionts. Our study demonstrated that microbial endosymbionts can affect the pathogenicity of Acanthamoeba; however, further research is required to clarify the exact pattern of symbiosis, in order to modify treatment protocol.

Fate of internalized Campylobacter jejuni and Mycobacterium avium from encysted and excysted Acanthamoeba polyphaga.

Association of the water- and foodborne pathogen Campylobacter jejuni with free-living Acanthamoeba spp. trophozoites enhances C. jejuni survival and resistance to biocides and starvation. When facing less than optimal environmental conditions, however, the Acanthamoeba spp. host can temporarily transform from trophozoite to cyst and back to trophozoite, calling the survival of the internalized symbiont and resulting public health risk into question. Studies investigating internalized C. jejuni survival after A. castellanii trophozoite transformation have neither been able to detect its presence inside the Acanthamoeba cyst after encystation nor to confirm its presence upon excystation of trophozoites through culture-based techniques. The purpose of this study was to detect C. jejuni and Mycobacterium avium recovered from A. polyphaga trophozoites after co-culture and induction of trophozoite encystation using three different encystation methods (Neff's medium, McMillen's medium and refrigeration), as well as after cyst excystation. Internalized M. avium was used as a positive control, since studies have consistently detected the organism after co-culture and after host excystation. Concentrations of C. jejuni in A. polyphaga trophozoites were 4.5 × 105 CFU/ml, but it was not detected by PCR or culture post-encystation. This supports the hypothesis that C. jejuni may be digested during encystation of the amoebae. M. avium was recovered at a mean concentration of 1.9 × 104 from co-cultured trophozoites and 4.4 × 101 CFU/ml after excystation. The results also suggest that M. avium recovery post-excystation was statistically significantly different based on which encystation method was used, ranging from 1.3 × 101 for Neff's medium to 5.4 × 101 CFU/ml for refrigeration. No M. avium was recovered from A. polyphaga cysts when trophozoites were encysted by McMillen's medium. Since C. jejuni internalized in cysts would be more likely to survive harsh environmental conditions and disinfection, a better understanding of potential symbioses between free-living amoebae and campylobacters in drinking water distribution systems and food processing environments is needed to protect public health. Future co-culture experiments examining survival of internalized C. jejuni should carefully consider the encystation media used, and include molecular detection tools to falsify the hypothesis that C. jejuni may be present in a viable but not culturable state.

Survival of Mycobacterium leprae and association with Acanthamoeba from environmental samples in the inhabitant areas of active leprosy cases: A cross sectional study from endemic pockets of Purulia, West Bengal.

BACKGROUND: Mycobacterium leprae being an obligate intracellular parasite cannot be cultured in any artificial culture media but it has been shown to reside in wild armadillos in North America. Many studies suggested that M. leprae could be found in the environment and may have a role in continuing transmission of the disease. The exact role of the environment in the transmission dynamics is still speculative. The present study was undertaken to find out the presence of viable M. leprae around patients' environment like soil and water and association of free living pathogenic protozoa, Acanthamoeba which might play an important role in transmission of the disease. METHODS: Seven hundred soil and 400 water samples were collected from the surroundings of the houses of leprosy patients from endemic villages. Two hundred soil and 80 water samples were also collected from the surroundings of normal inhabitants from non-endemic villages as controls. These samples were screened for the presence of M. leprae and Acanthamoeba using DNA PCR. RNA was extracted from the PCR positive samples and Reverse Transcriptase - PCR targeting 16S rRNA gene region was performed for detection of viable M. leprae. RESULTS: We observed high PCR positivity in soil samples (218 out of 700; 31%) and water samples (73 out of 400; 18%). These samples when further screened for viability, it was observed that 106 soil samples (15% of total) and 34 water samples (8% of total) showed presence of 16S rRNA. We observed 18.3% of soil and 20.5% of water samples were PCR positive for Acanthamoeba. Soil samples from the control area, where no active leprosy case resided in the last 5 years, showed PCR positivity in 4 samples (2%) for M. leprae DNA in only soil samples with all water samples being negative. RT-PCR for all PCR positive soil samples was negative. Of the 106 soil samples positive for M. leprae RT-PCR, 30 samples were also positive for Acanthamoeba whereas out of 112 M. leprae RT-PCR negative but PCR positive samples only 10 samples were Acanthamoeba positive showing association of viability with presence of Acanthamoeba (p = .0021). Similarly, for water samples also, association of M. leprae viability with presence of Acanthamoeba was seen (p = .0009). CONCLUSION: This study suggests that the surrounding environment (soil and water) of leprosy patients contain viable M. leprae and the viability has association with Acanthamoeba which may provide a protective niche for M. leprae. This could play an important role in the focal transmission of the disease.