Bacteriostatic effect of Echeveria extracts on diarrheagenic E. coli pathotypes and non-cytotoxicity on human Caco-2 cells.

INTRODUCTION: Diarrheagenic Escherichia coli pathotypes are important aetiological agents of diarrhoeal illness among children from less developed areas, worldwide. Diarrheagenic E. coli pathotypes strains are increasingly becoming drug resistant, thus effective and accessible therapeutic alternatives are required for their treatment; herbal extracts may be a potential alternative. AIMS: to evaluate Echeveria craigiana, E. kimnachii, and E. subrigida methanol extracts antibacterial effect on six diarrheagenic E. coli reference strains and on human colorectal adenocarcinoma cells viability and cytokine production. METHODOLOGY: Diarrheagenic E. coli pathotypes reference strains: typical enteropathogenic E2348/69, enterotoxigenic H10407, enterohaemorrhagic O157:H7/EDL933, enteroinvasive E11, diffusely adherent C18451-A, and enteroaggregative 042 E. coli. E craigiana, E. kimnachii, and E. subrigida leaves, collected at Sinaloa, Mexico, were freeze-dried and macerated in methanol solvent. Antibacterial activity was determined by a novel method developed in our laboratory, bacterial oxygen consumption by polarographic oxygen electrode technique and membrane integrity by two methods (live/dead and protein leakage assays). Colorectal adenocarcinoma cells viability by MTT assay and cytokine production using a Cytometric Bead Array kit. RESULTS: Extracts concentrations of 100 µg/mL and 5-hour incubation, reduced more than 93% the growth of all diarrheagenic E. coli pathotypes tested strains and significantly decreased bacterial oxygen consumption, like bacteriostatic antibiotics. After 24-hour incubation methanol extracts had a differential antibacterial effect on each diarrheagenic E. coli pathotypes strain. Echeveria extracts did not have any effect on viability and cytokine production of colorectal adenocarcinoma cells. CONCLUSIONS: Echeveria methanol extracts have a bacteriostatic effect on all diarrheagenic E. coli pathotypes strains, thus potentially they could be used as antibacterial agents on diarrheagenic E. coli pathotypes-contaminated products and on patients with diarrheagenic E. coli pathotypes infections.

Vitamin A supplementation modifies the association between mucosal innate and adaptive immune responses and resolution of enteric pathogen infections.

BACKGROUND: The efficacy of vitamin A supplementation on diarrheal disease morbidity may reflect the divergent effects that supplementation has on pathogen-specific immune responses and pathogen-specific outcomes. OBJECTIVE: We examined how vitamin A supplementation modified associations between gut-cytokine immune responses and the resolution of different diarrheal pathogen infections. DESIGN: Stools collected from 127 Mexican children who were 5-15 mo old and enrolled in a randomized, placebo-controlled vitamin A supplementation trial were screened for enteropathogenic Escherichia coli (EPEC), enterotoxigenic E. coli (ETEC), and Giardia lamblia. Fecal concentrations of interleukin (IL)-6, IL-8, IL-4, IL-5, IL-10, monocyte chemoattractant protein 1 (MCP-1), tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ) were measured by using an enzyme-linked immunosorbent assay. Hazard models that incorporated categorized cytokine variables (ie, nondetectable, less than the median of detectable concentrations, and at least the median of detectable concentrations) were fit to the length of pathogen infections stratified by treatment group. RESULTS: Vitamin A-supplemented children with fecal MCP-1 or IL-8 concentrations less than the median of detectable concentrations and IL-10 concentrations of at least median concentrations had longer durations of EPEC infection than did children in the placebo group. In supplemented children, detectable fecal TNF-α or IL-6 concentrations were associated with shorter ETEC infection durations, whereas MCP-1 concentrations of at least the median were associated with longer infection durations. Children in this group who had IL-4, IL-5, or IFN-γ concentrations of at least median detectable concentrations had shorter durations of G. lamblia infection. CONCLUSION: The effect of supplementation on associations between fecal cytokine concentrations and pathogen infection resolution depends on the role of inflammatory immune responses in resolving specific pathogen infections.

Associations between mucosal innate and adaptive immune responses and resolution of diarrheal pathogen infections.

The identification of immune response mechanisms that contribute to the control of diarrheal disease in developing countries remains an important priority. We addressed the role of fecal chemokines and cytokines in the resolution of diarrheal Escherichia coli and Giardia lamblia infections. Stools collected from 127 Mexican children 5 to 15 months of age enrolled in a randomized, double-blind, placebo-controlled, vitamin A supplementation trial were screened for enteropathogenic Escherichia coli (EPEC), enterotoxigenic E. coli (ETEC), and Giardia lamblia. Fecal concentrations of tumor necrosis factor alpha (TNF-alpha), monocyte chemoattractant protein-1 (MCP-1), interleukin-4 (IL-4), IL-5, IL-6, IL-8, IL-10, and interferon-gamma (IFN-gamma) were determined. Hazard models incorporating cytokine variables were fit to durations of asymptomatic and symptomatic pathogen infections, controlling for treatment group. Increased levels of TNF-alpha and IL-6 were associated with decreased durations of EPEC infection and increased ETEC durations. Increased IL-4 and IFN-gamma levels were associated with decreased and increased durations, respectively, of both EPEC and ETEC infections. Increased IL-10 levels were associated with increased and decreased durations of asymptomatic and symptomatic EPEC infections, respectively, and increased durations of both asymptomatic and symptomatic ETEC infections. Increased levels of MCP-1, IFN-gamma, IL-4, and IL-5 were associated with increased G. lamblia infection duration, while increased IL-8 levels were associated with decreased durations. Differences in proinflammatory and Treg cytokine levels are associated with differences in the resolution of inflammatory and noninflammatory pathogen infections.

Impact of vitamin A on selected gastrointestinal pathogen infections and associated diarrheal episodes among children in Mexico City, Mexico.

BACKGROUND: The overall effect of vitamin A supplementation on diarrheal disease in community trials may result from its effect on specific diarrheal pathogens. METHODS: We conducted a placebo-controlled, double-blind trial of the prophylactic effect of vitamin A on gastrointestinal pathogen infections and clinical symptoms among 188 children in Mexico City, Mexico, from January 1998 to May 1999. Children 6-15 months of age were randomly assigned to receive either a vitamin A supplement (for children <12 months of age, 20,000 international units [IU] of retinol; for children > or =12 months of age, 45,000 IU of retinol) every 2 months or a placebo and were followed for up to 15 months. Stool samples, collected semimonthly, were screened for enteropathogenic Escherichia coli (EPEC), enterotoxigenic E. coli (ETEC), Shiga toxin-producing E. coli (STEC), enteroinvasive E. coli (EIEC), and Giardia lamblia. RESULTS: Vitamin A supplementation reduced the prevalence of EPEC infections (rate ratio [RR], 0.52 [95% confidence interval {CI}, 0.23-0.86]) and led to shorter durations of both EPEC and ETEC infections. Supplementation also reduced the prevalence of EPEC-associated diarrhea (RR, 0.41 [95% CI, 0.16-1.00]), EPEC-associated fever (RR, 0.15 [95% CI, 0.02-0.98]), and G. lamblia-associated fever (RR, 0.27 [95% CI, 0.13-0.80]). Finally, children who received vitamin A supplementation had shorter durations of EPEC-associated diarrhea than did children who did not receive supplementation but had longer durations of G. lamblia-associated diarrhea. CONCLUSIONS: These results suggest that the effect of vitamin A supplementation on clinical outcomes may be pathogen dependent.

Vitamin A supplementation reduces the monocyte chemoattractant protein-1 intestinal immune response of Mexican children.

The impact of vitamin A supplementation on childhood diarrhea may be determined by the regulatory effect supplementation has on the mucosal immune response in the gut. Previous studies have not addressed the impact of vitamin A supplementation on the production of monocyte chemoattractant protein 1 (MCP-1), an essential chemokine involved in pathogen-specific mucosal immune response. Fecal MCP-1 concentrations, determined by an enzyme-linked immuno absorption assay, were compared among 127 Mexican children 5-15 mo of age randomized to receive a vitamin A supplement (<12 mo of age, 20,000 IU of retinol; > or =12 mo, 45,000 iu) every 2 mo or a placebo as part of a larger vitamin A supplementation trial. Stools collected during the summer months were screened for MCP-1 and gastrointestinal pathogens. Values of MCP-1 were categorized into 3 levels (nondetectable, or =median). Multinomial logistic regression models were used to determine whether vitamin A-supplemented children had different categorical values of MCP-1 compared with children in the placebo group. Differences in categorical values were also analyzed stratified by gastrointestinal pathogen infections and by diarrheal symptoms. Overall, children who received the vitamin A supplement had reduced fecal concentrations of MCP-1 compared with children in the placebo group (median pg/mg protein +/- interquartile range: 284.88 +/- 885.35 vs. 403.39 +/- 913.16; odds ratio 0.64, 95% CI 0.42-97, P = 0.03). Vitamin A supplemented children infected with enteropathogenic Escherichia coli (EPEC) had reduced MCP-1 levels (odds ratio = 0.38, 95% CI 0.18-0.80) compared with children in the placebo group. Among children not infected with Ascaris lumbricoides vitamin A supplemented children had reduced MCP-1 levels (OR = 0.62, 95% CI 0.41-0.94). These findings suggest that vitamin A has an anti-inflammatory effect in the gastrointestinal tract by reducing MCP-1 concentrations.

The effect of vitamin A supplementation on the intestinal immune response in Mexican children is modified by pathogen infections and diarrhea.

Vitamin A supplementation has consistently reduced infant mortality and the severity of pathogen-induced diarrhea. The mechanism by which vitamin A modulates the mucosal immune response to produce these effects remains poorly defined. To address this issue, stools collected during the summer months from 127 Mexican children 5-15 mo old enrolled in a larger, randomized, double-blind, placebo-controlled, vitamin A supplementation trial were screened for interleukin (IL)-4, IL-6, interferon-gamma (IFN-gamma), and gastrointestinal pathogens. Fecal cytokine values were categorized into 3 levels (undetectable, or =median). Multinomial regression models were used to determine the probability that vitamin A-supplemented children had higher categorical values of a cytokine than children in the placebo group. Differences in categorical values were also analyzed after stratification by gastrointestinal pathogen infections and diarrheal symptoms. Overall, fecal cytokine categorical levels did not differ between children randomized to the 2 arms. Vitamin A-supplemented children infected with enteropathogenic E. coli (EPEC) had reduced IL-4 and IFN-gamma levels [odds ratio (OR) = 0.3, 95% CI 0.13-0.67 and OR = 0.34, 95% CI 0.14-0.83, respectively] compared with children in the placebo group. Vitamin A-supplemented children had increased IL-4 levels when infected with A. lumbricoides (OR = 12.06, 95% CI 0.95-153.85). In contrast, IL-4 levels increased (OR = 2.14, 95% CI 0.94-4.87) and IFN-gamma levels decreased (OR = 0.51, 95% CI 0.26-0.99) among vitamin A-supplemented children with diarrhea compared with children in the placebo group. These findings suggest that the regulation of the mucosal immune response by vitamin A may depend on the type of enteric pathogen infecting the child and the presence of clinical symptoms.