Entamoeba histolytica Interaction with Enteropathogenic Escherichia coli Increases Parasite Virulence and Inflammation in Amebiasis.

Epidemiological studies suggest frequent association of enteropathogenic bacteria with Entamoeba histolytica during symptomatic infection. In this study, we sought to determine if the interaction with enteropathogenic (EPEC) or nonpathogenic Escherichia coli (strain DH5α) could modify the virulence of E. histolytica to cause disease in animal models of amebiasis. In vitro studies showed a 2-fold increase in CaCo2 monolayer destruction when E. histolytica interacted with EPEC but not with E. coli DH5α for 2.5 h. This was associated with increased E. histolytica proteolytic activity as revealed by zymogram analysis and degradation of the E. histolytica CP-A1/5 (EhCP-A1/5) peptide substrate Z-Arg-Arg-pNC and EhCP4 substrate Z-Val-Val-Arg-AMC. Additionally, E. histolytica-EPEC interaction increased EhCP-A1, -A2, -A4, and -A5, Hgl, Apa, and Cox-1 mRNA expression. Despite the marked upregulation of E. histolytica virulence factors, nonsignificant macroscopic differences in amebic liver abscess development were observed at early stages in hamsters inoculated with either E. histolytica-EPEC or E. histolytica-E. coli DH5α. Histopathology of livers of E. histolytica-EPEC-inoculated animals revealed foci of acute inflammation 3 h postinoculation that progressively increased, producing large inflammatory reactions, ischemia, and necrosis with high expression of il-1ß, ifn-γ, and tnf-α proinflammatory cytokine genes compared with that in livers of E. histolytica-E. coli DH5α-inoculated animals. In closed colonic loops from mice, intense inflammation was observed with E. histolytica-EPEC manifested by downregulation of Math1 mRNA with a corresponding increase in the expression of Muc2 mucin and proinflammatory cytokine genes il-6, il-12, and mcp-1 These results demonstrate that E. histolytica/EPEC interaction enhanced the expression and production of key molecules associated with E. histolytica virulence, critical in pathogenesis and progression of disease.

Common Polymorphisms Linked to Obesity and Cardiovascular Disease in Europeans and Asians are Associated with Type 2 Diabetes in Mexican Mestizos.

Background and objectives: Type 2 diabetes (T2D) is a major problem of public health in Mexico. We investigated the influence of five polymorphisms, previously associated with obesity and cardiovascular disease in Europeans and Asians, on T2D in Mexican Mestizos. Materials and Methods: A total of 1358 subjects from 30 to 85 years old were genotyped for five loci: CXCL12 rs501120; CDNK2A/B rs1333049; HNF-1α rs2259816; FTO rs9939609; and LEP rs7799039. We used logistic regressions to test the effect of each locus on T2D in two case⁻control groups with obesity and without obesity. Also, linear regression models on glucose and glycated hemoglobin (HbA1c) were carried out on the whole sample, adjusted by age, gender, and body mass index. Results: The CXCL12 rs501120 C allele (OR = 1.96, p = 0.02), the FTO rs9939609 A allele (OR = 2.20, p = 0.04) and the LEP rs7799039 A allele (OR = 0.6, p = 0.03) were significantly associated with T2D in obesity case⁻control group. No significant association was found in the non-obesity case⁻control group. The linear regression model showed that CDNK2A/B rs1333049 C allele (ß = 0.4, p = 0.03) and FTO rs9939609 A allele (ß = 0.5, p = 0.03), were significantly associated with HbA1c, but no association was found among the loci with the glucose levels. Conclusions: Polymorphisms previously linked with obesity and cardiovascular events were also associated with T2D and high levels of HbA1c. Furthermore, we must point at the fact that this is the first report where polymorphisms CXCL12 rs501120 and LEP rs7799039 are associated with T2D in subjects with obesity.

Escherichia coli enterotoxigénica y enteroagregativa: prevalencia, patogénesis y modelos múridos.

Enterotoxigenic (ETEC) and enteroaggregative Escherichia coli (EAEC) pathotypes are important etiological agents causative of diarrhea in children younger than 5 years of age in Mexico and in developing countries, where they cause numerous deaths. Both have been associated with delayed growth in children and are the main causative agents of traveler's diarrhea. The pathogenesis of both bacteria starts by adhering to the intestinal epithelium by means of fimbriae, called colonization factors in human ETEC isolates and aggregative adherence fimbriae in EAEC isolates. Once ETEC adheres to the enterocyte, it produces one or both of its toxins and induces the secretion of chloride and sodium ions and water into the intestinal lumen, producing its characteristic watery diarrhea. EAEC binds to the intestinal epithelium forming a biofilm, induces the production of mucus, releases its toxins and promotes inflammation. EAEC and ETEC infection models with wild-type C57BL/6 and CD40 ligand-deficient mice (with intact microbiota), respectively, revealed that undernutrition and low-zinc diet increases EAEC infection, causing growth retardation, and that ETEC colonizes, persists and induces local and systemic humoral immune response.

Los patotipos de Escherichia coli enterotoxigénica (ETEC) y enteroagregativa (EAEC) son importantes agentes etiológicos causantes de diarrea en niños menores de cinco años de México y países en desarrollo, en quienes causan numerosas muertes. Ambos se han asociado con retraso en el crecimiento infantil y son los principales agentes causales de la "diarrea del viajero". La patogénesis de ambas bacterias se inicia cuando estas se adhieren al epitelio intestinal mediante fimbrias, denominadas factores de colonización en las cepas ETEC aisladas de humano y fimbrias de adherencia agregativa en las cepas de EAEC. Una vez que ETEC se adhiere al enterocito produce una o ambas de sus toxinas e induce la secreción de iones de cloruro, sodio y agua al lumen intestinal, produciendo su característica diarrea acusa. EAEC se une al epitelio intestinal formando una biopelícula, induce la producción de moco, libera sus toxinas y promueve inflamación. Modelos de infección de EAEC y ETEC con ratones C57BL/6 silvestres y deficientes del ligando de CD40 (con microbiotas intactas), respectivamente, revelaron que la desnutrición y la dieta baja en cinc incrementan la infección de EAEC causando retraso en el crecimiento y que ETEC coloniza, persiste e induce respuesta inmune humoral local y sistémica.

Differential virome composition and richness between children's diarrheagenic stools kept at ultra-low temperatures for long-term.

INTRODUCTION: Diarrhoeal illness is the second cause of morbidity/mortality among children from less-developed regions worldwide. Nonetheless, there is scarce information regarding their gut microbiome. AIM: Microbiome characterization, with an emphasis on the virome, of children's stools with diarrhoea, by a commercial microbiome array. METHODOLOGY: Nucleic acids extraction, optimised for viral identification, of stool samples from 20 Mexican children with diarrhoea (10 children < 2 and 10 ≥ 2-years-old), collected 16 years ago and kept at -70 °C, were analysed for the presence of viruses, bacteria, archaea, protozoa, and fungi species sequences. RESULTS: Only viral and bacterial species sequences were identified among children's stools. Most stool samples harboured species belonging to the bacteriophages (95%), anellovirus (60%), diarrhoeagenic viruses (40%), and non-human pathogens viruses (45% avian virus and 40% plant viruses) groups. Among the children's stools, virome inter-individual species composition was observed, even in presence of illness. The < 2-years-old children group has significantly higher viral richness (p = 0.01), conferred mainly by bacteriophages and diarrheagenic-viruses (p = 0.01) species, in comparison with the ≥ 2-years-old group. CONCLUSIONS: The virome of stools of children with diarrhoea revealed inter-individual viral species composition. Similarly, to the few virome studies in healthy young children, the bacteriophages group was the most abundant. A significantly higher viral richness, conferred by bacteriophages and diarrheagenic-viral species, was observed among < 2-years-old children in comparison with older children. Stools preserved at -70 °C for long term can successfully be used for microbiome studies.

Elevated levels of enteric IgA in an unimmunised mouse model of Hyper IgM syndrome derived from gut-associated secondary lymph organs even in the absence of germinal centres.

Introduction: Patients with Human Hyper IgM syndromes (HIGM) developed pulmonary and gastrointestinal infections since infancy and most patients have mutations in the CD40 ligand (CD40L) gene. Most HIGM patients compared to healthy subjects have higher/similar IgM and lower IgG, and IgA serum concentrations but gut antibody concentrations are unknown. CD40L on activated T-cells interacts with CD40 on B-cells, essential for the formation of germinal centres (GCs) inside secondary lymphoid organs (SLOs), where high-affinity antibodies, long-lived antibody-secreting plasma cells, and memory B-cells, are produced. C57BL6-CD40 ligand deficient mice (C57BL6-cd40l -/-), are a model of HIGM, because serum immunoglobulin concentrations parallel levels observed in HIGM patients and have higher faecal IgA concentrations. In mice, TGFß and other cytokines induce IgA production. Aims: To compare and evaluate B-cell populations and IgA-producing plasma cells in peritoneal lavage, non-gut-associated SLOs, spleen/inguinal lymph nodes (ILN), and gut-associated SLOs, mesenteric lymph nodes (MLN)/Peyer´s patches (PP) of unimmunised C57BL6-cd40l -/- and C57BL6-wild-type (WT) mice. Material and methods: Peritoneal lavages, spleens, ILN, MLN, and PP from 8-10 weeks old C57BL6-cd40l -/- and WT mice, were obtained. Organ cryosections were analysed by immunofluorescence and B-cell populations and IgA-positive plasma cell suspensions by flow cytometry. Results: In unimmunised WT mice, GCs were only observed in the gut-associated SLOs, but GCs were absent in all C57BL6-cd40l -/- SLOs. PP and MLN of C57BL6-cd40l -/- mice exhibited a significantly higher number of IgA-producing cells than WT mice. In the spleen and ILN of C57BL6-cd40l- /- mice IgA-producing cells significantly decreased, while IgM-positive plasma cells increased. C57BL6-cd40l -/- B-1 cells were more abundant in all analysed SLOs, whereas in WT mice most B-1 cells were contained within the peritoneal cavity. C57BL6-cd40l -/- B-cells in MLN expressed a higher TGFß receptor-1 than WT mice. Mouse strains small intestine microvilli (MV), have a similar frequency of IgA-positive cells. Discussion: Together our results confirm the role of PP and MLN as gut inductive sites, whose characteristic features are to initiate an IgA preferential immune response production in these anatomical sites even in the absence of GCs. IgA antibodies play a pivotal role in neutralising, eliminating, and regulating potential pathogens and microorganisms in the gut.

Burden and transmission of zoonotic foodborne disease in a rural community in Mexico.

BACKGROUND: The foodborne transmission and human health impact of Salmonella and Campylobacter infections have rarely been evaluated at the population level in highly endemic settings. METHODS: A prospective 15-month cohort study of 127 infants and 119 elderly people was combined with animal and food surveillance to determine the incidence and severity of Salmonella and Campylobacter gastroenteritis in a comparatively prosperous rural community in Mexico. RESULTS: Salmonella and Campylobacter were isolated in up to 75% and 57%, respectively, of raw retail meat and in up to 4.5% of ready-to-eat foods. Rates of acute gastroenteritis of any etiology in infants and elderly people were, respectively, 2.1 and 0.7 episodes per person per year. The annual incidence density rate of Salmonella gastroenteritis was 17.8 per 100 infants and 7.9 per 100 elderly people; the rate of Campylobacter gastroenteritis was 11.7 per 100 infants and 0 per 100 elderly people. Pulsed-field gel electrophoresis analysis yielded multiple clusters of human, meat, and/or animal Salmonella and Campylobacter isolates with indistinguishable patterns. On average, gastroenteritis episodes with these pathogens lasted 3 days in infants and 2 days in elderly people. Medical attention was sought in 44% of diarrheal episodes in infants and in 26% of diarrheal episodes in elderly people; none required hospitalization. Infants with multidrug-resistant Salmonella gastroenteritis had a higher frequency of bloody stools and medical visits (50% vs 11%; odds ratio, 8.5; P = .04) than those with more susceptible strains. CONCLUSIONS: In this relatively advantaged Mexican rural community, the human health impact of a food chain heavily contaminated with Salmonella and Campylobacter was of low magnitude.

Antimicrobial-resistant Campylobacter in the food chain in Mexico.

We describe prevalence and antimicrobial susceptibility results for thermophilic Campylobacter isolates collected from humans, food, and food-animals in an integrated food chain surveillance network in Mexico. From 2003 to 2006, stool samples were collected from children with diarrhea at state sentinel hospitals. Concurrently, fecal samples from asymptomatic children in kindergartens, as well as raw chicken, pork and beef from retail outlets, and food-animal intestines from slaughterhouses were all collected in 65 cities from four different states. C. jejuni was identified with a standardized hippurate test. Hippurate negative, indoxyl acetate positive isolates were classified as Campylobacter spp. Susceptibility testing was performed by agar dilution according to Clinical and Laboratory Standards Institute guidelines. A total of 1,259 C. jejuni and 1,797 Campylobacter spp. isolates were recovered from 11,811 samples. Chicken was significantly more contaminated for both intestinal samples (93.6%) and meat products (58.3%), compared with swine (71.4%)/pork (14.6%) samples, and cattle (25.1%)/beef (5.3%) samples (p<0.001). Campylobacter was recovered from 5.1% of children with diarrhea and from 3.2% of asymptomatic children. Chicken was significantly more likely to harbor ciprofloxacin-resistant C. jejuni (85.8%) than swine (62.5%, OR=3.6), cattle (39.8%, OR=9.3), or humans (58.2%, OR=4.4). No significant differences were found for ciprofloxacin-resistant Campylobacter spp. among food-animals, but the rate in food-animals was significantly higher than in humans (84% vs. 56.7%, OR=4.0). Swine was significantly more likely to harbor erythromycin-resistant C. jejuni (14.8%) than chicken (3.5%, OR=4.9), cattle (1.8%, OR=9.3), or humans (3.0%, OR=5.7), and was associated with higher rates of erythromycin-resistant Campylobacter spp. (41.9%) than chicken (10.5%, OR=6.1) and humans (11.9%, OR=5.3). The high resistance rates to ciprofloxacin preclude the use of fluoroquinolones for treatment of campylobacteriosis in Mexico. Our results emphasize the need for ongoing and integrated surveillance of antimicrobial usage and antimicrobial susceptibility in humans and animals.

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.

Isolation of Staphylococcus aureus, Uropathogenic Escherichia coli, and Nontuberculous Mycobacteria Strains from Pasteurized Cheeses and Unpasteurized Cream Sold at Traditional Open Markets in Mexico City.

ABSTRACT: Fresh cheeses and cream are important garnishes of traditional Mexican food, often purchased at street or itinerant open markets or tianguis. However, there is scarce information regarding the microbiological quality of cheeses and cream sold in tianguis. For 2 years, three dairy stalls from three tianguis in Mexico City were visited once each season, trading practices were registered, and 96 dairy products were purchased. In total 72 fresh pasteurized cheeses that were hand-cut to order (24 Panela, 24 Canasto, and 24 Doble Crema) and 24 unpasteurized Crema de Rancho samples were collected. All dairy products remained without refrigeration for 8 h. Based on the National Guidelines limits, 87.5% of cheeses and 8% of Crema de Rancho samples were of low microbiological quality, and 1 sample of each type of cheese and 3 samples of Crema de Rancho exceeded the guidelines limits for Staphylococcus aureus. All dairy products were negative for Salmonella, Listeria monocytogenes, and all diarrheagenic Escherichia coli pathotypes, including Shiga toxin-producing E. coli. Among the 96 dairy samples, the prevalence of uropathogenic E. coli (UPEC) and of mycobacteria strains were determined because food items contaminated with these strains have been associated with urinary tract infections and mycobacteriosis, respectively. UPEC strains were isolated from 43% of cut-to-order cheeses and 29% of Crema de Rancho samples. Nontuberculous mycobacteria (NTM) strains were identified in 12.5% of Doble Crema cheese samples and 21% of Crema de Rancho samples. From the eight NTM-positive samples, 10 strains were identified (3 strains of Mycolicibacterium fortuitum, 2 of Mycobacteroides abscessus, 2 of Mycobacteroides chelonae, 2 of Mycolicibacterium porcinum, and 1 of Mycolicibacterium rhodesiae). All produced biofilms, and 70% had sliding motility (both virulence traits). Trading practices of cut-to-order pasteurized cheeses and unpasteurized Crema de Rancho in tianguis increase the risk of microbiological contamination of these products, including with human pathogens, and their consumption may cause human illness.

A Novel Adult Murine Model of Typical Enteroaggregative Escherichia coli Infection Reveals Microbiota Dysbiosis, Mucus Secretion, and AAF/II-Mediated Expression and Localization of ß-Catenin and Expression of MUC1 in Ileum.

Typical enteroaggregative Escherichia coli (tEAEC) is a diarrheagenic E. coli pathotype associated with pediatric and traveler's diarrhea. Even without diarrhea, EAEC infections in children also lead to increased gut inflammation and growth shortfalls. EAEC strain's defining phenotype is the aggregative adherence pattern on epithelial cells attributable to the aggregative adherence fimbriae (AAF). EAEC only causes diarrhea in humans; therefore, not much is known of the exact intestinal region of infection and damage or its interactions with intestinal enterocytes in vivo and in situ. This study aimed to develop a new tEAEC mouse model of infection, characterize the microbiota of infected mice, and evaluate in situ the expression of host adherence and surface molecules triggering EAEC infection and the role of the EAEC AAF-II in adherence. Six-week-old C57BL/6 mice, without previous antibiotic treatment, were orally challenged with EAEC 042 strain or EAEC 042 AAF-II mutant (ΔAAF/II) strain, or DAEC-MXR strain (diffusely adherent E. coli clinical isolate), and with saline solution (control group). Paraffin sections of the colon and ileum were stained with H&E and periodic acid-Schiff. ZO-1, ß-catenin, MUC1, and bacteria were analyzed by immunofluorescence. EAEC-infected mice, in comparison with DAEC-MXR-infected and control mice, significantly lost weight during the first 3 days. After 7 days post-infection, mucus production was increased in the colon and ileum, ZO-1 localization remained unaltered, and morphological alterations were more pronounced in the ileum since increased expression and apical localization of ß-catenin in ileal enterocytes were observed. EAEC-infected mice developed dysbiosis 21 days post-infection. At 4 days post-infection, EAEC strain 042 formed a biofilm on ileal villi and increased the expression and apical localization of ß-catenin in ileal enterocytes; these effects were not seen in animals infected with the 042 ΔAAF/II strain. At 3 days post-infection, MUC1 expression on ileal enterocytes was mainly detectable among infected mice and colocalized with 042 strains on the enterocyte surface. We developed a novel mouse model of EAEC infection, which mimics human infection, not an illness, revealing that EAEC 042 exerts its pathogenic effects in the mouse ileum and causes dysbiosis. This model is a unique tool to unveil early molecular mechanisms of EAEC infection in vivo and in situ.

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.

Human caliciviruses detected in Mexican children admitted to hospital during 1998-2000, with severe acute gastroenteritis not due to other enteropathogens.

Few studies exist regarding the frequency of human caliciviruses as single etiologic agents in sporadic cases, or in outbreaks occurring in children hospitalized for acute gastroenteritis. In this study, a total of 1,129 children of <5 years of age and hospitalized due to acute diarrhea were enrolled from three main hospitals in Mexico City during a period of 3 years (March 1998 to December 2000). After analyzing all fecal samples for several enteropathogens, 396 stools that remained negative were further screened for human caliciviruses by RT-PCR using a primer set specific to norovirus and sapovirus. Human caliciviruses were detected in 5.6% (22/396) of the children. The minimum incidence rate for 1999 were 5.3% (7/132) for 1999 and 7.8% (13/167) for 2000, since only fecal specimens that tested negative to other enteric pathogens were examined. Positive samples were further characterized using specific GI and GII primers and sequencing. Norovirus GII was detected in 19/22 samples, most of them were GII/4, while sapovirus GI/2 was detected in one sample. Associations between the presence of human calicivirus and clinical and epidemiological data revealed that diarrhea occurred with a seasonal pattern, and that children hospitalized due to human calicivirus disease scored an average of 13 +/- 3.2 (SD) points on the Vesikari scale, which corresponded to severe episodes. These results highlight that human caliciviruses, by themselves, are enteropathogens of acute severe diarrhea among young Mexican children requiring hospitalization and that their detection is important in order to reduce the diagnosis gap.

New Insights Into DAEC and EAEC Pathogenesis and Phylogeny.

Diarrheagenic E. coli can be separated into six distinct pathotypes, with enteroaggregative (EAEC) and diffusely-adherent E. coli (DAEC) among the least characterized. To gain additional insights into these two pathotypes we performed whole genome sequencing of ten DAEC, nine EAEC strains, isolated from Mexican children with diarrhea, and one EAEC plus one commensal E. coli strains isolated from an adult with diarrhea and a healthy child, respectively. These genome sequences were compared to 85 E. coli genomes available in public databases. The EAEC and DAEC strains segregated into multiple different clades; however, six clades were heavily or exclusively comprised of EAEC and DAEC strains, suggesting a phylogenetic relationship between these two pathotypes. EAEC strains harbored the typical virulence factors under control of the activator AggR, but also several toxins, bacteriocins, and other virulence factors. DAEC strains harbored several iron-scavenging systems, toxins, adhesins, and complement resistance or Immune system evasion factors that suggest a pathogenic paradigm for this poorly understood pathotype. Several virulence factors for both EAEC and DAEC were associated with clinical presentations, not only suggesting the importance of these factors, but also potentially indicating opportunities for intervention. Our studies provide new insights into two distinct but related diarrheagenic organisms.

Association of diarrheagenic Escherichia coli Pathotypes with infection and diarrhea among Mexican children and association of atypical Enteropathogenic E. coli with acute diarrhea.

Seventy-six children < or =2 years old were prospectively followed for 1 year in a peri-urban community of Mexico City to determine asymptomatic infection and acute diarrhea associated with diarrheagenic Escherichia coli pathotypes (DEPs). By use of a pathogen-specific multiplex PCR, DEPs were sought in 795 stool samples, of which 125 (16%) were positive for DEP; of these, 4 represented shedding episodes and 4 parasite coinfections. Most single-DEP infections (85/117) were asymptomatic (P < 0.001), and of the 32 DEP diarrhea episodes, 41% were associated with atypical enteropathogenic E. coli (aEPEC), 37.5% with enterotoxigenic E. coli, 9% with typical EPEC, 9% with enteroinvasive E. coli, and 3% with Shiga toxin-producing E. coli strains. Among the 76 children, 54 had at least one stool positive for DEP, of which 23 experienced a DEP-associated diarrhea episode. In the last group of children, DEP infection was significantly associated with a diarrhea episode (relative risk [RR] = 2.5; 95% confidence interval [CI], 1.79 to 3.57; P < 0.001), with ETEC (RR = 2.30; 95% CI, 1.49 to 3.54; P = 0.003) and aEPEC (RR = 1.92; 95% CI, 1.23 to 3.0; P = 0.019) being the pathotypes associated with diarrhea. aEPEC-associated diarrhea episodes were frequently in the <12-month age group (RR = 2.57; 95% CI, 1.05 to 6.27; P = 0.04). aEPEC infections were distributed all year round, but associated diarrheal episodes were identified from April to October, with a May-June peak (rainy season). Most ETEC infections and diarrhea episodes characteristically occurred during the summer (rainy season), with a diarrhea peak in August. Of all DEPs, only aEPEC was associated with acute diarrhea episodes lasting 7 to 12 days (P = 0.019). DEPs are important causes of community-acquired enteric infection and diarrhea in Mexican children.

Isolation of Mycobacterium mucogenicum from street-vended chili sauces: a potential source of human infection.

Recently human illnesses due to nontuberculous mycobacteria (NTM) have increased worldwide, but the sources of transmission have not been well established. Street-vended food is widely consumed in Mexico, and chili sauces are the most typical dressings for this food. Thus, we examined street-vended chili sauces as a possible source for NTM. Fifty-one street-vended chili sauces were collected in different areas of Mexico City during the spring of 2007. NTM were recovered from 6% (3 of 51) of samples, and in all cases the identified species was Mycobacterium mucogenicum. This mycobacterium has been associated with human illness; therefore, street-vended chili sauces are a potential source of NTM infection.

Draft Genome Sequence of Escherichia albertii Strain Mex-12/320a, Isolated from an Infant with Diarrhea and Harboring Virulence Genes Associated with Diarrheagenic Strains of Enteropathogenic Escherichia coli.

Escherichia albertii is an emerging human enteropathogen. We report the draft genome sequence of E. albertii strain Mex-12/320a, isolated from an infant with diarrhea. The presence of the pathogenic island O122/IE6 and the nleA gene, previously found in diarrheagenic enteropathogenic Escherichia coli strains, suggests that E. albertii may cause acute diarrhea.

Longitudinal Analysis of the Microbiological Quality of Raw Cow's Milk Samples Collected from Three Small Family Dairy Farms in Mexico over a 2-Year Period.

In Mexico, the total milk production that family dairy farms (FDF) contribute is ca. 35%, but this milk is not evaluated for microbiological quality. Forty percent of the milk and dairy products consumed by Mexicans is unpasteurized. In total, 24 raw cow's milk samples from three FDF (one sample per each season from each FDF for two sequent years) were characterized for the presence of food quality indicator organisms, Staphylococcus aureus, Salmonella enterica, Listeria monocytogenes, and Mycobacterium spp., by standard procedures. Escherichia coli presence was also evaluated by a direct count method and diarrheagenic E. coli (DEC) by molecular methods. On the basis of Mexican guidelines for raw milk entering production, 42% of samples exceeded the aerobic mesophilic bacteria limits. A total of 83% raw milk samples were positive for total coliforms, 54% for fecal coliforms, and 46% for E. coli. Forty-three E. coli isolates were selected and characterized for the presence of 11 DEC loci; of theses, 40 isolates were negative for all DEC loci, and 3 isolates, all collected from the same sample, were Shiga toxin 2 (stx2) positive and O157 antigen negative, and one stx2 isolate was resistant to 6 of the 16 antibiotics tested. None of the 24 raw milk samples were positive for Salmonella enterica, L. monocytogenes, or staphylococcal enterotoxin. S. aureus was isolated from nine samples, of which only three samples harbored resistant isolates. From three samples, four nontuberculous mycobacterial isolates were recovered (Mycobacteroides chelonae, Mycobacteroides porcinum, and two Mycobacteroides abscessus). All four isolates produced biofilm and had sliding motility, and three isolates (M. porcinum and two M. abscessus) were resistant to the two antibiotics tested (clarithromycin and linezolid). FDF provide raw milk to a large proportion of the Mexican population, but its consumption could be harmful to health, emphasizing the need to implement national microbiological quality guidelines for raw milk intended for direct human consumption.

Shared and organism-specific host responses to childhood diarrheal diseases revealed by whole blood transcript profiling.

Globally, diarrheal diseases are a leading cause of death in children under five and disproportionately affect children in developing countries. Children who contract diarrheal diseases are rarely screened to identify the etiologic agent due to time and cost considerations associated with pathogen-specific screening and hence pathogen-directed therapy is uncommon. The development of biomarkers to rapidly identify underlying pathogens could improve treatment options and clinical outcomes in childhood diarrheal diseases. Here, we perform RNA sequencing on blood samples collected from children evaluated in an emergency room setting with diarrheal disease where the pathogen(s) present are known. We determine host response gene signatures specific to Salmonella, Shigella and rotavirus, but not E. coli, infections that distinguish them from each other and from healthy controls. Specifically, we observed differential expression of genes related to chemokine receptors or inflammasome signaling in Shigella cases, such as CCR3, CXCR8, and NLRC4, and interferon response genes, such as IFI44 and OASL, in rotavirus cases. Our findings add insight into the host peripheral immune response to these pathogens, and suggest strategies and limitations for the use host response transcript signatures for diagnosing the etiologic agent of childhood diarrheal diseases.

Cytolethal distending toxin-producing Escherichia coli strains causing severe diarrhoea in young Mexican children.

Introduction. Cytolethal distending toxins (CDTs), encoded by cdt genes, have DNase activity leading to cellular and nuclear distension, resulting in irreversible cell cycle arrest and apoptosis of target cells. cdt-positive Escherichia coli strains have been isolated from children with diarrhoea. There is, however, scant information on the prevalence and clinical presentation of diarrhoeal disease caused by these strains. Furthermore, toxin production of cdt-positive strains is rarely confirmed. We report five young children with diarrhoea caused by CDT-producing E. coli in whom stools were negative for other bacterial or enteric pathogens. Case presentation. On admission to hospital, all children presented watery diarrhoea with high stool output (range 7-20 stools/24 h); five had fever of 38 °C or more and four presented vomiting. Dehydration was present in four patients, one of whom had hypovolaemic shock; one child also presented hyponatraemia and hypokalaemia. In two children, cdt-positive strains were classified as typical and atypical enteropathogenic E. coli, and the remaining three harboured cdt-positive strains that did not belong to any diarrhoeagenic pathogroup. One cdt-positive strain from each case was characterized by a CDT cytotoxic assay and a cdt type-specific PCR. All strains produced the characteristic cellular intoxication due to CDT. Two strains carried the cdt-I, one cdt-III, one cdt-IV, and one concurrently had cdt-I, cdt-II and cdt-III genes. Conclusion. Our results suggest that CDT-producing E. coli strains are an infrequent, albeit significant, cause of severe diarrhoeal illness in children. Future research should measure the true burden of cdt-positive E. coli diarrhoea among children.