Gut microbiome communication with bone marrow regulates susceptibility to amebiasis.

The microbiome provides resistance to infection. However, the underlying mechanisms are poorly understood. We demonstrate that colonization with the intestinal bacterium Clostridium scindens protects from Entamoeba histolytica colitis via innate immunity. Introduction of C. scindens into the gut microbiota epigenetically altered and expanded bone marrow granulocyte-monocyte progenitors (GMPs) and resulted in increased intestinal neutrophils with subsequent challenge with E. histolytica. Introduction of C. scindens alone was sufficient to expand GMPs in gnotobiotic mice. Adoptive transfer of bone marrow from C. scindens-colonized mice into naive mice protected against amebic colitis and increased intestinal neutrophils. Children without E. histolytica diarrhea also had a higher abundance of Lachnoclostridia. Lachnoclostridia C. scindens can metabolize the bile salt cholate, so we measured deoxycholate and discovered that it was increased in the sera of C. scindens-colonized specific pathogen-free and gnotobiotic mice, as well as in children protected from amebiasis. Administration of deoxycholate alone increased GMPs and provided protection from amebiasis. We elucidated a mechanism by which C. scindens and the microbially metabolized bile salt deoxycholic acid alter hematopoietic precursors and provide innate protection from later infection with E. histolytica.

The delicate balance between Entamoeba histolytica, mucus and microbiota.

Entamoeba histolytica (Eh) is a protozoan parasite of humans that colonizes the outer colonic mucus layer. Under conditions not fully understood, Eh breaches innate host defenses and invades the intestinal mucosa-causing amebic colitis and liver abscess. In asymptomatic infection, Eh interacts with and feeds on resident microbiota that forms biofilms on the outer colonic mucus layer. Despite the close association between Eh and commensal microbiota, we still lack basic knowledge on whether microbiota and/or their metabolites influence Eh virulence traits critical in disease pathogenesis. In the pathogenesis of intestinal amebiasis, Eh overcomes the protective mucus layer using a combination of mucinase/glycosidase and potent mucus secretagogue activity. In this addendum, we discuss the interconnected role of a healthy mucus barrier and the role commensal microbiota play in shaping innate host defense against Eh-induced pro-inflammatory and secretory responses critical in disease pathogenesis.

Learning from the research on amebiasis and gut microbiome: Is stimulation by gut flora essential for effective neutrophil mediated protection from external pathogens?

Amebiasis, caused by intestinal infection with Entamoeba histolytica, is one of the leading causes of parasite infection-related mortality and morbidity globally. Although its pathogenesis, including determinant factors of infection outcome, remains unclear, recent clinical data indicate that the gut microbiome plays a role in determining the severity of amebiasis. Recently, we investigated the effects of the gut microbiome on neutrophil mediated protection from E. histolytica infection using a mouse model. We identified that surface expression of CXCR2 on neutrophils was diminished in mice with dysbiosis, which resulted in decreased neutrophil recruitment to the infection site, allowing more aggressive intestinal tissue damage by E. histolytica. Our results indicated that oxidase activity during E. histolytica infection was also diminished after dysbiosis, consistent with the results from prior research. Thus, the gut microbiome plays an important role in regulating neutrophil phenotype when fighting against external pathogens.

Genome-Wide Association Study Reveals Genetic Link between Diarrhea-Associated Entamoeba histolytica Infection and Inflammatory Bowel Disease.

Entamoeba histolytica is the etiologic agent of amebic dysentery, though clinical manifestation of infection is highly variable ranging from subclinical colonization to invasive disease. We hypothesize that host genetics contribute to the variable outcomes of E. histolytica infection; thus, we conducted a genome-wide association study (GWAS) in two independent birth cohorts of Bangladeshi infants monitored for susceptibility to E. histolytica disease in the first year of life. Children with at least one diarrheal episode positive for E. histolytica (cases) were compared to children with no detectable E. histolytica infection in the same time frame (controls). Meta-analyses under a fixed-effect inverse variance weighting model identified multiple variants in a region of chromosome 10 containing loci associated with symptomatic E. histolytica infection. An intergenic insertion between CREM and CCNY (rs58000832) achieved genome-wide significance (P value from meta-analysis [Pmeta] = 6.05 × 10-9), and each additional risk allele of rs58000832 conferred 2.42 increased odds of a diarrhea-associated E. histolytica infection. The most strongly associated single nucleotide polymorphism (SNP) within a gene was in an intron of CREM (rs58468612; Pmeta = 8.94 × 10-8), which has been implicated as a susceptibility locus for inflammatory bowel disease (IBD). Gene expression resources suggest associated loci are related to the lower expression of CREM Increased CREM expression is also observed in early E. histolytica infection. Further, CREM-/- mice were more susceptible to E. histolytica amebic colitis. These genetic associations reinforce the pathological similarities observed in gut inflammation between E. histolytica infection and IBD.IMPORTANCE Diarrhea is the second leading cause of death for children globally, causing 760,000 deaths each year in children less than 5 years old. Amebic dysentery contributes significantly to this burden, especially in developing countries. The identification of host factors that control or enable enteric pathogens has the potential to transform our understanding of disease predisposition, outcomes, and treatments. Our discovery of the transcriptional regulator cAMP-responsive element modulator (CREM) as a genetic modifier of susceptibility to amebic disease has implications for understanding the pathogenesis of other diarrheal infections. Further, emerging evidence for CREM in IBD susceptibility suggests that CREM is a critical regulator of enteric inflammation and may have broad therapeutic potential as a drug target across intestinal inflammatory diseases.

Microbiome-mediated neutrophil recruitment via CXCR2 and protection from amebic colitis.

The disease severity of Entamoeba histolytica infection ranges from asymptomatic to life-threatening. Recent human and animal data implicate the gut microbiome as a modifier of E. histolytica virulence. Here we have explored the association of the microbiome with susceptibility to amebiasis in infants and in the mouse model of amebic colitis. Dysbiosis occurred symptomatic E. histolytica infection in children, as evidenced by a lower Shannon diversity index of the gut microbiota. To test if dysbiosis was a cause of susceptibility, wild type C57BL/6 mice (which are innately resistant to E. histiolytica infection) were treated with antibiotics prior to cecal challenge with E. histolytica. Compared with untreated mice, antibiotic pre-treated mice had more severe colitis and delayed clearance of E. histolytica. Gut IL-25 and mucus protein Muc2, both shown to provide innate immunity in the mouse model of amebic colitis, were lower in antibiotic pre-treated mice. Moreover, dysbiotic mice had fewer cecal neutrophils and myeloperoxidase activity. Paradoxically, the neutrophil chemoattractant chemokines CXCL1 and CXCL2, as well as IL-1ß, were higher in the colon of mice with antibiotic-induced dysbiosis. Neutrophils from antibiotic pre-treated mice had diminished surface expression of the chemokine receptor CXCR2, potentially explaining their inability to migrate to the site of infection. Blockade of CXCR2 increased susceptibility of control non-antibiotic treated mice to amebiasis. In conclusion, dysbiosis increased the severity of amebic colitis due to decreased neutrophil recruitment to the gut, which was due in part to decreased surface expression on neutrophils of CXCR2.

Real-time analysis of gut flora in Entamoeba histolytica infected patients of Northern India.

BACKGROUND: Amebic dysentery is caused by the protozoan parasite Entamoeba histolytica and the ingestion of quadrinucleate cyst of E. histolytica from fecally contaminated food or water initiates infection. Excystation occurs in the lumen of small intestine, where motile and potentially invasive trophozoites germinate from cysts. The ability of trophozoites to interact and digest gut bacteria is apparently important for multiplication of the parasite and its pathogenicity; however the contribution of resident bacterial flora is not well understood. We quantified the population of Bacteroides, Bifidobacterium, Ruminococcus, Lactobacillus, Clostridium leptum subgroup, Clostridium coccoides subgroup, Eubacterium, Campylobacter, Methanobrevibacter smithii and Sulphur reducing bacteria using genus specific primers in healthy (N = 22) vs amebic patients (E. histolytica positive, N = 17) stool samples by Real-time PCR. RESULTS: Absolute quantification of Bacteroides (p = .001), Closrtridium coccoides subgroup (p = 0.002), Clostridium leptum subgroup (p = 0.0001), Lactobacillus (p = 0.037), Campylobacter (p = 0.0014) and Eubacterium (p = 0.038) show significant drop in their population however, significant increase in Bifdobacterium (p = 0.009) was observed where as the population of Ruminococcus (p = 0.33) remained unaltered in healthy vs amebic patients (E. histolytica positive). We also report high prevalence of nimE gene in stool samples of both healthy volunteers and amebic patients. No significant decrease in nimE gene copy number was observed before and after the treatment with antiamebic drug. CONCLUSIONS: Our results show significant alteration in predominant gut bacteria in E. histolytica infected individuals. The frequent episodes of intestinal amoebic dysentery thus result in depletion of few predominant genera in gut that may lead to poor digestion and absorption of food in intestine. It further disturbs the homeostasis between gut epithelium and bacterial flora. The decrease in beneficial bacterial population gives way to dysbiosis of gut bacteria which may contribute to final outcome of the disease. Increase in the copy number of nimE gene harboring bacteria in our population reflects possible decrease in the availability of metronidazole drug during treatment of amoebiasis.

Nuevos métodos para el diagnóstico microbiológico de la amebiasis y de otros protozoarios intestinales / New methods for the microbiological diagnosis of amebiasis and other intestinal protozoa

La Organización Mundial de la Salud (OMS) ubica a las enfermedades diarreicas en segundo lugar como causa de morbi-mortalidad de niños en países en vías de desarrollo, siendo las infecciones por protozoarios intestinales proporcionalmente una causa trascendente en dichas regiones. La mayoría de estos parásitos se transmiten por vía fecal-oral o por contacto interpersonal y exhiben ciclosde vida que, en general se desarrollan en dos estadios: el trofozoíto que coloniza el intestino y el quiste que es excretado con las heces y cuya rígida pared protectora, le confiere resistencia en el ambiente, permitiendo de este modo la propagación de la enfermedad. Entamoeba histolytica es uno de los agentes infecciosos de mayor distribución mundial, y es aproximadamente la causa de 100.000 muertes por año, siendo responsable de uno de los problemas de salud más serios en países en vías de desarrollo. Se reconocen al menos ocho amebas que podrían colonizar el intestino del hombre: E. histolytica, E. dispar, E. moshkovskii, E. coli, E. hartmanni, E. polecki, Iodamoeba butschlii y Endolimax nana. Aunque sólo E. histolytica es considerada como el agente etiológico de la Amebiosis. Su presentación clínica va desde la colonización asintomática, la disentería amebiana, hasta la propagación extraintestinal, originando abscesos en diversos órganos y tejidos. Por este motivo, la correcta identificación de E. histolytica en heces y tejidos y diferenciándola de otras amebas comensales y de otros protozoarios representa un desafío en la práctica médica, a que de ello dependerá el tratamiento y el pronóstico de laenfermedad.

Abstract: The World Health Organization (WHO) includes diarrhea a the second leading cause of morbidity and mortality of children in developing countries. Among these causes, infections by intestinal protozoan parasites represent an important percentage in any place of the world. Most of these parasites are transmitted by the fecal-oral route or by inter personal contact and exhibit simple life cycles, consisting in the disease-causing, proliferating trophozoites and the dormant, resistant cyst responsible for the transmission of the infection among susceptible hosts. Entamoeba histolytica is one of the most frequent of those parasites, causing about 100.000 deaths per year in developing countries, being one of the major health problems in areas where basic sanitation practices are inefficient. At least eight species of Entamoeba have been reported to infect the human large intestine: E. histolytica, E. dispar, E. moshkovskii, E. coli, E. hartmanni, E. polecki, odamoeba butschlii and Endolimax nana, although E. histolytica is the only one considered to cause pathology in humans. Clinical manifestation of this infection varies from asymptomatic infection to dysentery and extraintestinal invasion, producing abscesses in many tissues. Therefore, the correct identification of E. histolytica in stool and tissue samples and its differential diagnostic is an important challenge in parasitology because treatment and prognosis depend of the valid identification of this parasite.The health issue of protozoan intestinal infections, both in developed and developing parts of the world, is of such importance that it is clearly necessary the development of novel, better, cheap, and faster diagnostic methods. The incorporation of new approaches and technology to the efficient and sensitive detection of these infections is significantly relevant.

Role of cysteine proteinase of Entamoeba histolytica in target cell death.

The bacterial flora of the intestine plays an important role in the virulence caused by Entamoeba histolytica. Cysteine proteinase (CP), an amoebic virulence factor, plays a major role in host cell destruction. The mechanism of increased virulence following bacterial co-association is not understood. We studied CP of E. histolytica HM1:IMSS which was co-associated with Escherichia coli K12 strain pre-incubated with GalNAc or CP specific inhibitor E 64. Co-association of E. histolytica with bacteria enhanced CP activity 3-6-fold as assessed by azocasein assay and substrate gel electrophoresis showed bands at molecular weights of 28, 35 and 56 kDa. Northern and Western blot analysis showed increase in ehcp2 and ehcp5 gene expression. Trophozoites co-associated with E. coli showed greater cytotoxicity of BHK cells by a 51Cr release assay than trophozoites that had not been co-associated; this enhancement was abolished by E-64 treatment. The killing of BHK 21 targets by E. histolytica was characterized by DNA laddering which was not inhibited with E-64. GalNAc pre-incubation of trophozoites reduced cytotoxicity and DNA laddering, while E. coli co-associated E. histolytica showed smearing with faint laddering of BHK implicating both necrosis and apoptosis. Hence, bacterial co-association increases CP activity and CP gene expression and contributes to the necrosis of the target cell.

Biological aspects of a Brazilian strain of Entamoeba dispar.

A strain of Entamoeba dispar was characterized by clinical diagnosis, serological and electrophoretical isoenzyme analysis and by the polymorphism of a 482 bp genomic fragment analysis. The pathogenesis and virulence of this strain was investigated considering the experimental infection in hamster livers in association with the original intestinal microbiota. Liver lesions were observed in hamsters experimentally infected with trophozoites from xenic cultures, but not from the monoxenic cultures. Moreover, clones obtained from re-isolated strain Wil1R1 showed a distinct biological behavior. In fact, animals inoculated with Wil1R1ClB3 showed an intense acute inflammatory reaction with destructive focal hepatic lesions. These lesions were characterized as amebic abscesses. The association between bacteria and ameba has been fairly well studied because it affects the pathogenicity of the amebas and has important therapeutic implications. In this study, we demonstrated that E. dispar in association with the original microbiota is able to produce lesions in hamster liver in spite of its having been considered to be non-pathogenic in the hamster model. Based on these results we suggest that diagnosis of amebiasis needs to be made with more care and that clinical and therapeutical procedures need to be revised.

[Infectious etiology of dysenteric syndromes at the Principal Hospital of Dakar in 1999 and 2000]. / Etiologies infectieuses des syndromes dysentériques à l'Hôpital Principal de Dakar en 1999 et 2000.

Dysenteric syndromes are relatively frequent in tropical areas, due essentially to infectious etiologies, constituted by flimsy pathogenic agents outside and possibilities of laboratory investigations little available. The authors evaluated during two years (1990-2000) the results of 399 parasitological examinations and 419 bacteriological examinations concerning dysenteric syndromes admitted to the laboratory of "Hopital Principal de Dakar". The objective of these examination is to contribute to the management of cases. The patients are divided into two groups: the first group is constituted by children less than two years old hospitalised in the pediatric services, and the second group includes all the other patients. The parasitological studies schow that parasitic etiologies are almost non existent in the first group while in the second group, they are essentially represented by Entamoeba histolytic with 19.5% of prevalence. The bacteriological studies show 42.7% of positivity rate in the first group and 19.5% in the second group with a predominance of E. coli and Shigella dysenteriae. The prevalence or pathogenic agents associations is not neglectable: they represent 8% or positive results with a predominance of amoeba-Shigella association. These results confirm the necessity of a best case management during the preanalytic phase in order to improve the scores of positivity and the particularities of hospitalised children les than two years old, to when the frequency of observed cases is high with bacteriological etiologies essentially (E. coli EPEC+, Nosocomial bacteria).

Bacterium-assisted invasion of Entamoeba histolytica through human enteric epithelia in two-compartment chambers.

Entamoeba histolytica trophozoites initiate amebiasis by invasion into the enteric mucosa. It is the aim of our experiments to understand how bacteria and leukocytes act during amebic invasion through enteric cell layers. Cocultures were established in two-compartment chambers and studied by measurement of transepithelial electrical resistance (TER) and by histological examination. Trophozoites caused a decrease in TER that was followed by formation of holes in the enteric cell layer and transfilter migration of trophozoites. Phagocytosed bacteria activated trophozoites that opened the intracellular junctions and provided access for the invasion of bacteria. Leukocytes had no effect on the different steps of invasion of the trophozoites through the human enteric cell layers. We conclude that trophozoites, eventually assisted by enteric bacteria, disrupt enterocytic tight junctions before they open the enteric cell layer and invade through it.

Inhibitory activity of saccharomyces yeasts on the adhesion of Entamoeba histolytica trophozoites to human erythrocytes in vitro.

Adhesion to target cells represents the first step in infection by Entamoeba histolytica. Binding of axenic amoeba (HMI strain) to human red cells in vitro was employed as a model of the adhesion process. The influence of precontact of trophozoites with suspensions of live Saccharomyces boulardii yeasts, their fractions (membranes and yeast-content supernatant before and after filtration to eliminate the membrane) or yeast culture medium before and after fermentation was investigated. N-Acetylgalactosamine (GalNAC) was employed as the reference inhibitory sugar. The percentage of amoebae bearing red cells after pretreatment of amoebae with the various suspensions and derivates was determined. Adhesion was also evaluated by scanning electron microscopy (SEM). Pretreatment of amoebae with the live yeast suspension led to a significant reduction in the percentage of adhesion [32% vs 70% in the phosphate-buffered saline (PBS) control]. Reduced adhesion was also observed with the filtered and unfiltered supernatant of the yeast suspension homogenate [32% and 34%, respectively, vs 69% in the PBS control], yeast culture medium at the end of fermentation [49% vs 76% in the PBS control] and GalNAC [32% vs 72% in the PBS control]. SEM showed a decrease in the number of amoebae bearing red cells and a reduction in the number of red cells adhering to amoebae. We conclude that substances produced by the yeasts compete with red cells for adhesion sites on amoebae.

[Fatal transmural colitis caused by Entamoeba histolytica. Differential diagnosis of chronic inflammatory bowel disease and colon cancer]. / Entamoeba histolyticabetinget transmural colitis med letalt forløb. En differentialdiagnose til kronisk inflammatorisk tarmsygom og cancer coli.

Perforation of the colon is a rare but frequently fatal complication of amoebiasis. We report a case of a 53 year-old male, with no history of travel abroad, who was admitted to hospital with haematochezia. A tumor of the rectum was diagnosed clinically. Due to acute intestinal obstruction, laparotomy was performed, revealing multiple perforations of the large bowel and severe peritonitis leading to subtotal colectomy. The histological examinations revealed transmural amoebic colitis. The patient died due to multi-organ failure.