Ana Carolina Ramos Moreno

Etiology of childhood diarrhea in the northeast of Brazil: significant emergent diarrheal pathogens

In a study conducted in João Pessoa, northeast of Brazil, 2344 Escherichia coli isolated from 290 infants with diarrhea and 290 healthy matched controls were analyzed for virulence traits. Enteroaggregative E. coli (EAEC) was the most prevalent pathogen associated to acute diarrhea. Based on the results of colony blot hybridization, serotyping, and HEp-2 cell adherence assays, strains were separated in categories as typical enteropathogenic E. coli (EPEC) (1.7%), atypical EPEC (a-EPEC) (9.3%), EAEC (25%), enterotoxigenic E. coli (10%), and enteroinvasive E. coli (EIEC) (1.4%). No enterohemorrhagic E. coli strains were isolated. Other enteropathogens were found, including Salmonella (7.9%), Shigella spp. (4.1%), thermophilic Campylobacter spp. (2.4%), Giardia lamblia (9.3%), and Entamoeba histolytica (5.8%). All enteropathogens were associated with diarrhea (P < 0.01). However, the association was lower for EPEC and EIEC (P < 0.03). Different pathogens associated with diarrhea may have been changing in Brazil where EAEC and a-EPEC seem to be the most prevalent pathogens among them.

Mesenteric microcirculatory dysfunctions and translocation of indigenous bacteria in a rat model of strangulated small bowel obstruction

PRUPOSE Bacterial translocation has been shown to occur in critically ill patients after extensive trauma, shock, sepsis, or thermal injury. The present study investigates mesenteric microcirculatory dysfunctions, the bacterial translocation phenomenon, and hemodynamic/metabolic disturbances in a rat model of intestinal obstruction and ischemia. METHODS Anesthetized (pentobarbital 50 mg/kg, i.p.) male Wistar rats (250–350 g) were submitted to intestinal obstruction or laparotomy without intestinal obstruction (Sham) and were evaluated 24 hours later. Bacterial translocation was assessed by bacterial culture of the mesenteric lymph nodes (MLN), liver, spleen, and blood. Leukocyte-endothelial interactions in the mesenteric microcirculation were assessed by intravital microscopy, and P-selectin and intercellular adhesion molecule (ICAM)-1 expressions were quantified by immunohistochemistry. Hematocrit, blood gases, lactate, glucose, white blood cells, serum urea, creatinine, bilirubin, and hepatic enzymes were measured. RESULTS About 86% of intestinal obstruction rats presented positive cultures for E. coli in samples of the mesenteric lymph nodes, liver, and spleen, and 57% had positive hemocultures. In comparison to the Sham rats, intestinal obstruction induced neutrophilia and increased the number of rolling (~2-fold), adherent (~5-fold), and migrated leukocytes (~11-fold); this increase was accompanied by an increased expression of P-selectin (~2-fold) and intercellular adhesion molecule-1 (~2-fold) in the mesenteric microcirculation. Intestinal obstruction rats exhibited decreased PaCO2, alkalosis, hyperlactatemia, and hyperglycemia, and increased blood potassium, hepatic enzyme activity, serum urea, creatinine, and bilirubin. A high mortality rate was observed after intestinal obstruction (83% at 72 h vs. 0% in Sham rats). CONCLUSION Intestinal obstruction and ischemia in rats is a relevant model for the in vivo study of mesenteric microcirculatory dysfunction and the occurrence of bacterial translocation. This model parallels the events implicated in multiple organ dysfunction (MOD) and death.

Can the fliC PCR-Restriction Fragment Length Polymorphism Technique Replace Classic Serotyping Methods for Characterizing the H Antigen of Enterotoxigenic Escherichia coli Strains?

ABSTRACT In this study, we performed fliC PCR-restriction fragment length polymorphism (RFLP) to investigate whether this technique would be better than classic serotyping for the characterization of the H antigen in enterotoxigenic Escherichia coli (ETEC) strains. We showed that the fliC genes from ETEC strains can be characterized by restriction analysis of their polymorphism. Only one allele of the fliC gene from ETEC strains was found for each flagellar antigen, with the exception of H21. Nonmotile strains could also be characterized using this molecular technique. Moreover, determination of the somatic antigen was guided by the identification of the flagellar antigen from previously unknown serotypes of ETEC strains by PCR-RFLP, thus reducing the number of anti-antigen O sera used. The PCR-RFLP technique proved to be faster than classic serotyping for the characterization of the E. coli H antigen, taking 2 days to complete instead of the 7 or more days using classic serotyping. In conclusion, the H molecular typing for Enterobacteriaceae members may become an important epidemiological tool for the characterization of the H antigen of E. coli pathotypes. The PCR-RFLP technique is capable of guiding the determination of the H antigen and could partially replace seroagglutination. With the determination of the molecular profiles of alleles from strains obtained in epidemiological studies, new patterns will be described for ETEC strains or other E. coli pathotypes, thus permitting widespread use of this technique to characterize fliC genes and determine the H antigen of E. coli strains.In this study, we performed fliC PCR-restriction fragment length polymorphism (RFLP) to investigate whether this technique would be better than classic serotyping for the characterization of the H antigen in enterotoxigenic Escherichia coli (ETEC) strains. We showed that the fliC genes from ETEC strains can be characterized by restriction analysis of their polymorphism. Only one allele of the fliC gene from ETEC strains was found for each flagellar antigen, with the exception of H21. Nonmotile strains could also be characterized using this molecular technique. Moreover, determination of the somatic antigen was guided by the identification of the flagellar antigen from previously unknown serotypes of ETEC strains by PCR-RFLP, thus reducing the number of anti-antigen O sera used. The PCR-RFLP technique proved to be faster than classic serotyping for the characterization of the E. coli H antigen, taking 2 days to complete instead of the 7 or more days using classic serotyping. In conclusion, the H molecular typing for Enterobacteriaceae members may become an important epidemiological tool for the characterization of the H antigen of E. coli pathotypes. The PCR-RFLP technique is capable of guiding the determination of the H antigen and could partially replace seroagglutination. With the determination of the molecular profiles of alleles from strains obtained in epidemiological studies, new patterns will be described for ETEC strains or other E. coli pathotypes, thus permitting widespread use of this technique to characterize fliC genes and determine the H antigen of E. coli strains.

Prevalence of secreted autotransporter toxin gene among diffusely adhering Escherichia coli isolated from stools of children

In this report, we analyzed the prevalence of the sat gene in 336 Escherichia coli samples collected from stools of children with and without diarrhea in Brazil and in 100 uropathogenic E. coli strains. The results show a high correlation between diffusely adhering E. coli (DAEC) and the presence of sat (44%) in intestinal isolates. DAEC strain FBC114 expresses a 107-kDa protein, which showed 98% homology with Sat.

Enteroinvasive Escherichia coli vs. Shigella flexneri : how different patterns of gene expression affect virulence

Important features of the enteroinvasive Escherichia coli (EIEC) phenotype and gene expression likely to confer EIEC with a lower ability to cause disease than Shigella flexneri were described here for the first time. To confirm the lower pathogenicity of EIEC, we have analyzed the keratoconjunctivitis developed in guinea-pigs with EIEC or S. flexneri. Shigella flexneri induced a more pronounced proinflammatory response, whereas EIEC induced a mild form of the disease. EIEC showed a significantly less efficient cell-to-cell Caco-2 dissemination when compared with S. flexneri. Plaques formed by EIEC during intercellular spreading were four times smaller than those formed by S. flexneri. At the molecular level, the lower expression of virulence genes by EIEC during infection of Caco-2 cells highlighted the importance of effective gene transcription for bacterial pathogenicity.

HYPERTONIC SALINE SOLUTION REDUCES MESENTERIC MICROCIRCULATORY DYSFUNCTIONS AND BACTERIAL TRANSLOCATION IN A RAT MODEL OF STRANGULATED SMALL BOWEL OBSTRUCTION

ABSTRACT We examined the effects of hypertonic saline (HS) on inflammatory, metabolic variables, and bacterial translocation (BT) in rats submitted to intestinal obstruction and ischemia (IO). Male Wistar rats were submitted to IO and treated, 2 h thereafter, with lactated Ringer’s (LR) (4 mL/kg per 5 min, i.v.) or HS (7.5% NaCl, 4 mL/kg per 5 min, i.v.). Twenty-four hours after IO, rats were also submitted to enterectomy/enteroanastomosis to resection of necrotized small bowel. Leukocyte-endothelial interactions were investigated by intravital microscopy and the expression of P-selectin and intercellular adhesion molecule 1 by immunohistochemistry. Bacterial cultures of mesenteric lymph nodes, liver, spleen, and blood were used to evaluate BT. Levels of chemokines (cytokine-induced neutrophil chemoattractants 1 and 2), insulin, and corticosterone were determined by enzyme-linked immunosorbent assay. Intestinal histology, serum urea and creatinine levels, and hepatic enzymes activities were performed to evaluate local and remote damage. Relative to IO and LR-treated rats, which exhibited increases in the number of rolling (1.5-fold), adhered (3.5-fold) and migrated (9.0-fold) leukocytes, and increased expression of P-selectin (3-fold) and intercellular adhesion molecule 1 (3-fold) on mesenteric microcirculation, treatment with HS followed by enterectomy reduced leukocyte-endothelial interactions and expression of both adhesion molecules to values attained in sham rats. Serum chemokines were normalized after treatment with both solutions followed by enterectomy. Hypertonic saline–treated rats demonstrated a significant reduction in BT to 50% in liver and spleen samples and bacteremia (14%), compared with 82% of BT in liver and spleen samples of IO and LR-treated rats and bacteremia (57%). Local intestinal damage was attenuated, and renal and hepatic function preserved by treatment with HS followed by enterectomy. Survival rate increased to 86% up to 15 days. Data presented suggest that HS solution followed by enterectomy reduces mesenteric microcirculatory dysfunctions and BT, attenuating local and remote damage in a model of strangulated small bowel obstruction.

Expression of bacterial virulence factors and cytokines during in vitro macrophage infection by enteroinvasive Escherichia coli and Shigella flexneri: a comparative study

Enteroinvasive Escherichia coli (EIEC) and Shigella spp cause bacillary dysentery in humans by invading and multiplying within epithelial cells of the colonic mucosa. Although EIEC and Shigella share many genetic and biochemical similarities, the illness caused by Shigella is more severe. Thus, genomic and structure-function molecular studies on the biological interactions of these invasive enterobacteria with eukaryotic cells have focused on Shigella rather than EIEC. Here we comparatively studied the interactions of EIEC and of Shigella flexneri with cultured J774 macrophage-like cells. We evaluated several phenotypes: (i) bacterial escape from macrophages after phagocytosis, (ii) macrophage death induced by EIEC and S. flexneri, (iii) macrophage cytokine expression in response to infection and (iv) expression of plasmidial (pINV) virulence genes. The results showed that S. flexneri caused macrophage killing earlier and more intensely than EIEC. Both pathogens induced significant macrophage production of TNF, IL-1 and IL-10 after 7 h of infection. Transcription levels of the gene invasion plasmid antigen-C were lower in EIEC than in S. flexneri throughout the course of the infection; this could explain the diminished virulence of EIEC compared to S. flexneri.

The expanding roles of 1‐methyl‐tryptophan (1‐MT): in addition to inhibiting kynurenine production, 1‐MT activates the synthesis of melatonin in skin cells

Indoleamine 2,3‐dioxygenase 1 (IDO1), the rate‐limiting enzyme of tryptophan catabolism, has been strongly associated with the progression of malignancy and poor survival in melanoma patients. As a result, IDO1 is a leading target for interventions aimed at restoring melanoma immune surveillance. Here, in a scenario involving the tryptophan catabolism, we report that melatonin biosynthesis is driven by 1‐methyl‐tryptophan (1‐MT), a competitive inhibitor of IDO1, in human fibroblasts, melanocytes and melanoma cells. In addition to melatonin biosynthesis, 1‐MT induced the expression of tryptophan hydroxylase, arylalkylamine‐N‐acetyltransferase and hydroxyindole O‐methyltransferase mRNA in fibroblasts and melanocytes. We observed a great variability in the levels of IDO1 mRNA expression and kynurenine release between skin cells and melanoma cell lines in response to interferon‐γ, a classical IDO1 inducer. In this setting, melatonin was shown to downregulate kynurenine production. Furthermore, in a condition of low basal activity of IDO1, it was observed that 1‐MT, as well melatonin, inhibited the proliferation of human melanoma cells. Taken together, our results suggest that 1‐MT may serve as more than just a tool to disrupt tumor immune escape (via the inhibition of IDO1) because it was shown to act directly on the proliferation of human melanoma cells and induce melatonin biosynthesis in the tumor milieu. Moreover, 1‐MT‐mediated inhibition of IDO occurs in normal skin and melanoma cells, which addresses the possibility that all cells in the skin microenvironment can be targeted by 1‐MT. Our findings provide innovative approaches into understanding tumor therapy related to the control of tryptophan metabolism by 1‐MT.

Bacillus subtilis spores as adjuvants for DNA vaccines

Recently, Bacillus subtilis spores were shown to be endowed with strong adjuvant capacity when co-administered with purified antigenic proteins. In the present study we assessed whether spores possess adjuvant properties when combined with DNA vaccines. We showed that B. subtilis spores promoted the activation of dendritic cells in vitro and induced migration of pro-inflammatory cells after parenteral administration to mice. Likewise, co-administration of spores with a DNA vaccine encoding the human papillomavirus type 16 (HPV-16) E7 protein enhanced the activation of antigen-specific CD8(+) T cell responses in vivo. Mice immunized with the DNA vaccine admixed with spores presented a protective immunity increase to previously implanted tumor cells, capable of expressing HPV-16 oncoproteins. Finally, we observed that the adjuvant effect can vary accordingly to the number of co-administered spores which may be ascribed with the ability to induce. Collectively, the present results demonstrate for the first time that B. subtilis spores can also confer adjuvant effects to DNA vaccines.

Biosynthesis of N,N-dimethyltryptamine (DMT) in a melanoma cell line and its metabolization by peroxidases.

Tryptophan (TRP) is essential for many physiological processes, and its metabolism changes in some diseases such as infection and cancer. The most studied aspects of TRP metabolism are the kynurenine and serotonin pathways. A minor metabolic route, tryptamine and N,N-dimethyltryptamine (DMT) biosynthesis, has received far less attention, probably because of the very low amounts of these compounds detected only in some tissues, which has led them to be collectively considered as trace amines. In a previous study, we showed a metabolic interrelationship for TRP in melanoma cell lines. Here, we identified DMT and N,N-dimethyl-N-formyl-kynuramine (DMFK) in the supernatant of cultured SK-Mel-147 cells. Furthermore, when we added DMT to the cell culture, we found hydroxy-DMT (OH-DMT) and indole acetic acid (IAA) in the cell supernatant at 24 h. We found that SK-Mel-147 cells expressed mRNA for myeloperoxidase (MPO) and also had peroxidase activity. We further found that DMT oxidation was catalyzed by peroxidases. DMT oxidation by horseradish peroxidase, H2O2 and MPO from PMA-activated neutrophils produced DMFK, N,N-dimethyl-kynuramine (DMK) and OH-DMT. Oxidation of DMT by peroxidases apparently uses the common peroxidase cycle involving the native enzyme, compound I and compound II. In conclusion, this study describes a possible alternative metabolic pathway for DMT involving peroxidases that has not previously been described in humans and identifies DMT and metabolites in a melanoma cell line. The extension of these findings to other cell types and the biological effects of DMT and its metabolites on cell proliferation and function are key questions for future studies.