Enteropathogenic Escherichia coli modulates the virulence and pathogenicity of Entamoeba dispar.

Amoebiasis is a disease caused by Entamoeba histolytica, affecting the large intestine of humans and occasionally leading to extra-intestinal lesions. Entamoeba dispar is another amoeba species considered commensal, although it has been identified in patients presenting with dysenteric and nondysenteric colitis, as well as amoebic liver abscess. Amoebic virulence factors are essential for the invasion and development of lesions. There is evidence showing that the association of enterobacteria with trophozoites contributes to increased gene expression of amoebic virulence factors. Enteropathogenic Escherichia coli is an important bacterium causing diarrhea, with high incidence rates in the world population, allowing it to interact with Entamoeba sp. in the same host. In this context, this study aims to evaluate the influence of enteropathogenic Escherichia coli on ACFN and ADO Entamoeba dispar strains by quantifying the gene expression of virulence factors, including galactose/N-acetyl-D-galactosamine-binding lectin, cysteine proteinase 2, and amoebapores A and C. Additionally, the study assesses the progression and morphological aspect of amoebic liver abscess and the profile of inflammatory cells. Our results demonstrated that the interaction between EPEC and ACFN Entamoeba dispar strains was able to increase the gene expression of virulence factors, as well as the lesion area and the activity of the inflammatory infiltrate. However, the association with the ADO strain did not influence the gene expression of virulence factors. Together, our findings indicate that the interaction between EPEC, ACFN, and ADO Entamoeba dispar strains resulted in differences in vitro and in vivo gene expression of Gal/GalNAc-binding lectin and CP2, in enzymatic activities of MPO, NAG, and EPO, and consequently, in the ability to cause lesions.

Co-infection by Salmonella enterica subsp. Enterica serovar typhimurium and Entamoeba dispar pathogenic strains enhances colitis and the expression of amoebic virulence factors.

Amebiasis is the most severe protozoan infection affecting the human intestine, and the second leading cause of death among parasitic diseases. The mechanisms of amoebic virulence factors acquisition are poorly understood, and there are few studies showing the interaction between Entamoeba dispar and bacteria. Salmonella enterica subsp. enterica serovar typhimurium is also a common cause of gastroenteritis in humans. Considering the high rates of amebiasis and salmonellosis, it is possible that these diseases may co-exist in the human intestine, leading to co-infection. Due to the scarcity of studies showing the influence of enteropathogenic bacteria on amoebic virulence, our research group proposed to evaluate the impact of S. typhimurium on E. dispar trophozoites. We assessed whether co-infection of S. typhimurium and E. dispar can change the progression of amoebic colitis, and the inflammatory response profile in the caecum mucosa, using a co-infection experimental model in rats. In vitro assays was used to investigate whether S. typhimurium induces changes in amoebic virulence phenotype. In the present work, we found that S. typhimurium co-infection exacerbates amoebic colitis and intestinal inflammation. The in vitro association of S. typhimurium and E. dispar trophozoites contributed to increase the expression of amoebic virulence factors. Also, we demonstrated, for the first time, the cysteine proteinase 5 expression in E. dispar MCR, VEJ and ADO strains, isolated in Brazil. Together, our results show that S. typhimurium and E. dispar co-infection worsens amoebic colitis, possibly by increasing the expression of amoebic virulence factors.

Probiotic Propionibacterium freudenreichii requires SlpB protein to mitigate mucositis induced by chemotherapy.

Propionibacterium freudenreichii CIRM-BIA 129 (P. freudenreichii wild type, WT) is a probiotic bacterium, which exerts immunomodulatory effects. This strain possesses extractable surface proteins, including SlpB, which are involved in anti-inflammatory effect and in adhesion to epithelial cells. We decided to investigate the impact of slpB gene mutation on immunomodulation in vitro and in vivo. In an in vitro assay, P. freudenreichii WT reduced expression of IL-8 (p<0.0001) and TNF-α (p<0.0001) cytokines in LPS-stimulated HT-29 cells. P. freudenreichii ΔslpB, lacking the SlpB protein, failed to do so. Subsequently, both strains were investigated in vivo in a 5-FU-induced mucositis mice model. Mucositis is a common side effect of cytotoxic chemotherapy with 5-FU, characterized by mucosal injury, inflammation, diarrhea, and weight loss. The WT strain prevented weight loss, reduced inflammation and consequently histopathological scores. Furthermore, it regulated key markers, including Claudin-1 (cld1, p<0.0005) and IL-17a (Il17a, p<0.0001) genes, as well as IL-12 (p<0.0001) and IL-1ß (p<0.0429) cytokines levels. Mutant strain displayed opposite regulatory effect on cld1 expression and on IL-12 levels. This work emphasizes the importance of SlpB in P. freudenreichii ability to reduce mucositis inflammation. It opens perspectives for the development of probiotic products to decrease side effects of chemotherapy using GRAS bacteria with immunomodulatory surface protein properties.

Selection of starter cultures for the production of sour cassava starch in a pilot-scale fermentation process

ABSTRACT Sour cassava starch (Polvilho azedo) is obtained from a spontaneous fermentation conducted by microorganisms from raw materials and fermentation tanks. This product is traditionally used in the baking industry for the manufacture of biscuits and Brazilian cheese breads. However, the end of fermentation is evaluated empirically, and the process occurs without standardization, which results in products of inconsistent quality. Predominant microbiota from a cassava flour manufacturer was isolated in order to select starter cultures for the production of sour cassava starch in a pilot-scale fermentation process. Lactic acid bacteria and yeasts were isolated, enumerated and grouped by Restriction Fragment Length Polymorphism, and PCR fingerprinting, respectively. One isolate of each molecular profile was identified by sequencing of the rRNA gene. LAB were prevalent throughout the entire process. Lactobacillus brevis (21.5%), which produced the highest values of acidity, and Lactobacillus plantarum (13.9%) were among the most frequent species. Pichia scutulata (52.2%) was the prevalent yeast and showed amylolytic activity. The aforementioned species were tested as single and mixed starter cultures in a pilot-scale fermentation process for 28 days. L. plantarum exhibited better performance as a starter culture, which suggests its potential for the production of sour cassava starch.

Porcine stomachs with and without gastric ulcer differ in Lactobacillus load and strain characteristics.

Although Lactobacillus species are recognized as normal inhabitants of porcine gastric mucosa, the association of these bacteria with health status or gastric ulcer disease has never been considered. We investigated the bacterial load of Lactobacillus isolated from the antrum, corpus, and pars esophagea of stomachs with (n = 13) and without (n = 10) ulcer of the pars esophagea of slaughtered pigs. We also evaluated in vitro antagonistic properties against typical pathogens of strains isolated from stomachs without ulcer. To quantify Lactobacillus, gastric mucosa samples obtained with 5 mm biopsy punches were smeared on MRS agar and colonies were counted after 48 h of incubation under anaerobic conditions. The score of Lactobacillus was significantly greater in the antrum and corpus of stomachs without ulcer (P < 0.001 for both) when compared with stomachs with ulcer. Fingerprint profiles, obtained by repetitive sequence-based PCR using (GTG)5 primers, showed that the isolates were highly diverse. The reduction of Lactobacillus load in porcine stomachs may be a contributing factor for gastric ulcer. Strains isolated from healthy stomachs, which showed a wide spectrum of antagonistic activity against pathogens, may be viewed as an untapped source of bacteria with potential beneficial properties that deserve to be further investigated.

Identification of Leptospira serovars by RFLP of the RNA polymerase beta subunit gene (rpoB)

Leptospires are usually classified by methods based on DNA-DNA hybridization and the conventional cross-agglutination absorption test, which uses polyclonal antibodies against lipopolysaccharides. In this study, the amplification of the rpoB gene, which encodes the beta-subunit of RNA polymerase, was used as an alternative tool to identify Leptospira. DNA extracts from sixty-eight serovars were obtained, and the hypervariable region located between 1990 and 2500-bp in the rpoB gene was amplified by polymerase chain reaction (PCR). The 600-bp amplicons of the rpoB gene were digested with the restriction endonucleases TaqI, Tru1I, Sau3AI and MslI, and the restriction fragments were separated by 6% polyacrylamide gel electrophoresis. Thirty-five fragment patters were obtained from the combined data of restriction fragment length polymorphism (PCR-RFLP) analysis and used to infer the phylogenetic relationships among the Leptospira species and serovars. The species assignments obtained were in full agreement with the established taxonomic classifications. Twenty-two serovars were effectively identified based on differences in their molecular profiles. However, the other 46 serovars remained clustered in groups that included more than one serovar of different species. This study demonstrates the value of RFLP analysis of PCR-amplified rpoB as an initial method for identifying Leptospira species and serovars.

Stable genetic transformation of malaria parasites by homologous recombination

A transformaçäo genética de diferentes estágios dos parasitas da malária já é possível atualmente. Um conjunto crescente de marcadores seletivos estäo sendo desenvolvidos para permitir uma manipulaçäo genética mais completa de Plasmodium. "Gene targeting", que permite romper um gene ou introduzir alterações sutis na seqüência de um gene via recombinaçäo homóloga, é uma nova tecnologia usada no estudo de estrutura-funçäo de antígenos in vivo. A criaçäo de parasitas recombinantes carreando mutações pontuais em sítios conservados de TRAP de esporozoitas de P. berghei é usado como protótipo desta nova tecnologia.