Modifying effects of 2,4-D and Glyphosate exposures on gut-liver-adipose tissue axis of diet-induced non-alcoholic fatty liver disease in mice.

Nonalcoholic fatty liver disease (NAFLD), which is linked to western diet (WD) intake, affects 30% of the world's population and involves the crosstalk of liver steatosis, hypertrophy/inflammation of adipose tissue and deregulation of gut microbiome. Glyphosate and 2,4-D are some of the most applied herbicides worldwide, and their roles in NAFLD have not been investigated. Thus, the present study evaluated whether glyphosate and 2,4-D, in single or mixed exposure, alter WD-induced NAFLD in a mouse model. Male C57Bl/6 mice (n = 10/group) received a fat (30% lard, 0.02% cholesterol), and sucrose-rich diet (20%) and high sugar solution (23.1 and 18.9 g/L of fructose and glucose) for 6 months. Simultaneously, animals received glyphosate (0.05 or 5 mg/kg/day), 2,4-D (0.02 or 2 mg/kg/day), or their combination (0.05 +0.02 or 5 +2 mg/kg/day) by intragastrical administration (5 ×/week). Doses were based on the Acceptable Daily Intake (ADIs) or No Observed Adverse Effect Level (NOAEL) levels. Herbicide exposures featured differential responses. WD-induced obesity, hypercholesterolemia, and hyperglycemia remained unaltered. Compared to the group receiving only WD, only the concomitant exposure to WD and 2,4-D (2 mg) enhanced the percentage of mice with moderate/severe hepatic inflammation, CD68 macrophage infiltration, and malondialdehyde levels in the liver. In line, this herbicide modulated immune response- (including Cd4, C8b, Cd28, Cxcr3, Cxcr6) and oxidative stress-related (such as Gsta1, Gsta2, Gsta4, Gstm1, Gstm2, Gstm3, Gstm4, Nqo1, Gpx2) genes in the hepatic transcriptome analysis. This exposure also enriched pro-inflammatory Deferribacteres phylum in fecal microbiome. In general, the herbicide mixtures did not feature the same effects attributed to 2,4-D isolated exposure. Our findings indicate that 2,4-D, at a dose within the toxicological limits, was able to induce disturbances in mainly at the liver and gut axes involved in NAFLD development in male mice.

Indole-3-carbinol and chlorogenic acid combination modulates gut microbiome and attenuates nonalcoholic steatohepatitis in a murine model.

Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease worldwide, affecting almost 32% of the population and ranging from simple steatosis to nonalcoholic steatohepatitis (NASH). Recent findings indicate that the fast-growing prevalence of NAFLD might be linked to adherence to a Westernized diet (WD), mostly composed of fat/sugar-enriched foods. The WD has been reportedly targeted as a potential driver of gut-liver axis unbalance, suggesting a major role in NASH. On the other hand, bioactive food compounds feature as a potential chemopreventive strategy against NASH, due to their beneficial effects (i.e, anti-inflammatory/oxidant activity and modulation of gut microbiome). Brassicaceae vegetables are known for their high amount of isothiocyanates and polyphenols, as indole-3-carbinol (I3C) and chlorogenic acid (CGA). Thus, we sought to assess the effects of human relevant doses of I3C and CGA isolated or in combination (5/125 mg/Kg of body weight, respectively) on a diet/chemical-induced murine model of NASH. I3C + CGA oral treatment diminished NAFLD activity score (NAS) (p < 0.0001), as well as alleviated the hepatic lipid (p = 0.0011) accumulation, prevented hepatic stellate cell (HSC) activation (p < 0.0001), and subsequent fibrosis (p < 0.0001). The combination also reduced the number of both hepatic CD68-positive macrophages (p < 0.0001) and cleaved caspase-3 hepatocytes (p < 0.0001) and diminished the malondialdehyde levels (p = 0.0155). Additionally, the combination of I3C + CGA restored the relative abundance of Alistipes (p = 0.0299), Allobaculum (p = 0.0014), Bacteroides (p = 0.0046), and Odoribacter (p = 0.0030) bacteria genera on the gut microbiome. Taken together, these findings show that the combination of I3C + CGA at populational-relevant ingestion, rather than the I3C or CGA alone, was able to modulate gut microbiome and attenuate NASH in this hybrid model mouse.

Western diet-induced mouse model of non-alcoholic fatty liver disease associated with metabolic outcomes: Features of gut microbiome-liver-adipose tissue axis.

OBJECTIVES: Non-alcoholic fatty liver disease (NAFLD) has a growing epidemiologic and economic burden. It is associated with Western diet (WD) patterns, and its pathogenesis involves metabolic disorders (obesity, dyslipidemia, hyperglycemia, and diabetes) and gut dysbiosis, features that are usually neglected or not reproduced by most animal models. Thus, we established a 6-mo WD-induced NAFLD mouse model associated with metabolic disorder, investigating its main features at the gut microbiome-liver-adipose tissue axis, also evaluating the correlations of gut dysbiosis to the other disease outcomes. METHODS: Male C57 BL6 mice received a high-fat (30% lard and 0.2% cholesterol, ∼57% calories) and sucrose-rich (20%) chow, and a high-sugar solution (23.1 and 18.9 g/L of D-fructose and D-glucose) for 6 mo. RESULTS: The model featured high serum cholesterol levels, glucose intolerance, and hyperinsulinemia. WD intervention resulted in extensive macro/microvesicular liver steatosis and pericellular fibrosis-resembling human disease-accompanied by hepatic stellate cell activation and CD68+ macrophage infiltration, increased protein levels of proinflammatory p65-nuclear factor-κB, interleukin-6 and tumor necrosis factor-α, with decreased antioxidant regulator Nrf2. Mice showed clear obesity with adipocyte hypertrophy, and CD68+macrophage/mast cell infiltration in adipose tissue while a reduction in number of goblet cells was also observed in the small intestine. Moreover, the pyrosequencing of the 16 S ribosomal RNA of gut cecal content showed decreased bacterial diversity, enriched Firmicutes and Proteobacteria, decreased Bacteroidetes and Fusobacteria, and increased ratio of Firmicutes to Bacteroidetes. Bacteroidetes and Bacteroides had the highest number of significant correlations with liver-adipose tissue axis outcomes. In silico analysis of gut microbiome in NAFLD obese patients revealed a depletion in Bacteroides, which also correlated to disease outcomes. CONCLUSION: This mice model gathered suitable phenotypical alterations in gut-liver-adipose tissue axis that resembled NAFLD associated with metabolic disorders in humans and may be considered for preclinical investigation.

Role of the YehD fimbriae in the virulence-associated properties of enteroaggregative Escherichia coli.

The interaction of enteroaggregative Escherichia coli (EAEC) strains with the colonic gut mucosa is characterized by the ability of the bacteria to form robust biofilms, to bind mucin, and induce a local inflammatory response. These events are mediated by a repertoire of five different aggregative adherence fimbriae variants (AAF/I-V) typically encoded on virulence plasmids. In this study, we report the production in EAEC strains of a new YehD fimbriae (YDF), which is encoded by the chromosomal gene cluster yehABCD, also present in most E. coli strains. Immuno-labelling of EAEC strain 042 with anti-AAF/II and anti-YDF antibodies demonstrated the presence of both AAF/II and YDF on the bacterial surface. We investigated the role of YDF in cell adherence, biofilm formation, colonization of spinach leaves, and induction of pro-inflammatory cytokines release. To this aim, we constructed yehD deletion mutants in different EAEC backgrounds (strains 17-2, 042, 55989, C1010, 278-1, J7) each harbouring one of the five AAFs. The effect of the YDF mutation was strain dependent and AAF independent as the lack of YDF had a different impact on the phenotypes manifested by the different EAECs tested. Expression of the yehABCD operon in a E. coli K12 ORN172 showed that YDF is important for biofilm formation but not for adherence to HeLa cells. Lastly, screening of pro-inflammatory cytokines in supernatants of Caco-2 cells infected with EAEC strains 042 and J7 and their isogenic ΔyehD mutants showed that these mutants were significantly defective in release of IL-8 and TNF-α. This study contributes to the understanding of the complex and diverse mechanisms of adherence of EAEC strains and identifies a new potential target for preventive measures of gastrointestinal illness caused by EAEC and other E. coli pathogroups.

Role of the YehD fimbriae in the virulence-associated properties of enteroaggregative Escherichia coli

The interaction of enteroaggregative Escherichia coli (EAEC) strains with the colonic gut mucosa is characterized by the ability of the bacteria to form robust biofilms, to bind mucin, and induce a local inflammatory response. These events are mediated by a repertoire of five different aggregative adherence fimbriae variants (AAF/I-V) typically encoded on virulence plasmids. In this study, we report the production in EAEC strains of a new YehD fimbriae (YDF), which is encoded by the chromosomal gene cluster yehABCD, also present in most E. coli strains. Immuno-labelling of EAEC strain 042 with anti-AAF/II and anti-YDF antibodies demonstrated the presence of both AAF/II and YDF on the bacterial surface. We investigated the role of YDF in cell adherence, biofilm formation, colonization of spinach leaves, and induction of pro-inflammatory cytokines release. To this aim, we constructed yehD deletion mutants in different EAEC backgrounds (strains 17-2, 042, 55989, C1010, 278-1, J7) each harbouring one of the five AAFs. The effect of the YDF mutation was strain dependent and AAF independent as the lack of YDF had a different impact on the phenotypes manifested by the different EAECs tested. Expression of the yehABCD operon in a E. coli K12 ORN172 showed that YDF is important for biofilm formation but not for adherence to HeLa cells. Lastly, screening of pro-inflammatory cytokines in supernatants of Caco-2 cells infected with EAEC strains 042 and J7 and their isogenic ΔyehD mutants showed that these mutants were significantly defective in release of IL-8 and TNF-α. This study contributes to the understanding of the complex and diverse mechanisms of adherence of EAEC strains and identifies a new potential target for preventive measures of gastrointestinal illness caused by EAEC and other E. coli pathogroups.

Gut dysbiosis in mice genetically selected for low antibody production.

BACKGROUND: Dysbiosis is linked to the cause of several human diseases, many of which having an immunity related component. This work investigated whether mice genetically selected for low or high antibody production display differences in intestinal bacterial communities, and consisted in the comparison of fecal 16SV6-V8 rDNA PCR amplicons resolved by temperature gradient gel electrophoresis (TGGE) of five each of low (LIII) and high (HIII) antibody producing mice. 16SV6 rDNA amplicons of 2 mice from each line were sequenced. RESULTS: LIII mice were grouped in a single TGGE cluster, displayed a low α-diversity, and were distinguished by low Firmicutes/Bacteroidetes ratio. CONCLUSION: The results suggest that genetically driven low antibody production in mice is associated with gut dysbiosis.

Gut dysbiosis in mice genetically selected for low antibody production

Background: Dysbiosis is linked to the cause of several human diseases, many of which having an immunity related component. This work investigated whether mice genetically selected for low or high antibody production display differences in intestinal bacterial communities, and consisted in the comparison of fecal 16SV6-V8 rDNA PCR amplicons resolved by temperature gradient gel electrophoresis (TGGE) of five each of low (LIII) and high (HIII) antibody producing mice. 16SV6 rDNA amplicons of 2 mice from each line were sequenced. Results: LIII mice were grouped in a single TGGE cluster, displayed a low a-diversity, and were distinguished by low Firmicutes/Bacteroidetes ratio. Conclusion: The results suggest that genetically driven low antibody production in mice is associated with gut dysbiosis.

Draft Genome Sequence of Shiga Toxin-Producing Escherichia coli Strain D92/09.

Escherichia coli is suspected to be involved with Crohn's disease. Adherence and invasion to epithelial cells are properties commonly observed in these bacteria. Here, we present a draft genome sequence of E. coli D92/09, a multidrug-resistant strain, which besides showing these properties produces Shiga cytotoxin-1 and possibly other toxins.

Escherichia coli from Crohn's disease patient displays virulence features of enteroinvasive (EIEC), enterohemorragic (EHEC), and enteroaggregative (EAEC) pathotypes.

BACKGROUND: Escherichia coli is a normal inhabitant of the gut which upon acquiring virulence factors becomes potentially able to cause diseases. Although E. coli population augments in Crohn's disease (CD), the reason of this proliferation is not yet clear. CD associated E. coli shows features of extraintestinal pathogenic categories (ExPEC), and eventually the ability to invade cultured epithelial cells, a property observed among diarrheagenic E. coli (DEC). In this work, data on the characterization of an E. coli isolate from a CD patient reveal that, besides invasiveness, CD associated E. coli may harbor other typical DEC markers, namely those defining enterohemorragic (EHEC) and enteroaggregative (EAEC) pathotypes. RESULTS: The studied strain, detected both in an ileum biopsy and stools, belonged to the B2 E. coli reference collection (EcoR) phylogroup and harbored the intimin, Shiga cytotoxin 1, and AggR transcriptional activator encoding genes (eae, stx1, aggR, respectively); displayed aggregative adherence to Hep-2 cells and an ability to enter Caco-2 cells four times as high as that of EIEC reference strain and half of invasiveness of AIEC LF82. It was able to enter and replicate in J774 macrophages with invasiveness 85 times as high as that of LF82, but with only one sixth of the intracellular proliferation ability of the later. Extracellular products with cytotoxic activity on Vero cells were detected in strain's cultures. Preliminary analysis indicated similarity of this strain's genome with that of O104:H4/2011C-3493. METHODS: Following its isolation from a resected CD patient, the strain was characterized by in vitro adhesion and invasion assays to Hep-2, invasion to Caco-2 cells and to J774 macrophages and tested for the ability to form biofilm and to produce Shiga cytotoxins. PCRs were carried out to identify virulence genetic markers and for EcoR phylogrouping. The strain's genome was sequenced by means of Ion torrent PGM platform. CONCLUSION: The detection, in a CD patient, of an E. coli combining virulence features of multiple DEC pathotypes seems not only to stress the relevance of E. coli to CD etiopathogenesis but also to indicate the existence of new and potentially more virulent strains putatively associated with this disease.

Pathotypes and probiotics: response to a commentary on the detection of a Shiga toxin producing Escherichia coli in a Crohn's disease patient.

A recent report on the detection in a Crohn's disease (CD) patient of an adherent and invasive Shiga toxin producing Escherichia coli (STEC) (Gut pathogens 2015, 7:2) prompted a commentary expressing some skepticism on the significance of the paper findings (Gut pathogens 2015, 7:15). Besides focusing on recurrent issues concerning the difficulties in defining a pathogen, the opinion considers recent data demonstrating the presence of virulence factors in a commercial probiotic. In response to the commentary's observations, additional information on the described STEC strain, as well as a short discussion on CD associated E. coli are presented here.

Mucosa-associated but not luminal Escherichia coli is augmented in Crohn's disease and ulcerative colitis.

BACKGROUND: Escherichia coli is believed to participate in the etiology of Crohn's disease (CD) and possibly of ulcerative colitis (UC), due at least in part to the observed rise in the number of these bacteria in the gut microbiota of CD and UC patients. Nevertheless, it is not fully understood whether this quantitative variation occurs equally throughout the mucosal and luminal spaces of the gut. To assess this question, stools and mucosa biopsies from distinct intestinal sites were cultured aiming at determining their E. coli concentration. The cultures were additionally screened for the presence of some virulence genes of pathogenic E. coli. RESULTS: Analyses of clinical materials from 14 controls (38 biopsies and 14 stools samples), 11 CD (25 biopsies and 11 stools samples) and 7 UC patients (18 biopsies and 7 stools samples) indicated no significant variation in the number of E. coli present in stools, but a rise of at least one log10 CFU/mg in biopsies from the ileum of CD patients and the sigmoid and rectum of CD and UC patients. The cultures were screened for the presence of E. coli attaching and effacing (eae), invasion plasmid antigen H (ipaH), aggregative adherence transcriptional activator (aggR), Shiga cytotoxins (stx), and heat labile enterotoxin (elt) and the following serine proteases autotransporters of Enterobacteriaceae (SPATE) genes: plasmid encoded toxin (pet), secreted autotransporter toxin (sat), Shigella extracellular protein (sepA), protein involved in intestinal colonization (pic) and Shigella IgA-like protease homolog (sigA). Six of the 10 genes screened were detected in the total of samples investigated: aggR, eae, pet, sat, sepA and sigA. No difference in the prevalence of any of these markers was observed in cultures from different clinical materials or groups of patients. METHODS: Bacterial quantitation was carried out following cultures of diluted samples suspensions in MacConkey agar, Wilkins Chalgren agar for anaerobes, E. coli/coliform chromocult agar, and blood agar. Screening for E. coli virulence genes was performed by multiplex PCR of DNA purified from total MacConkey undiluted broth cultures. CONCLUSION: In CD and UC patients only the mucosa associated population of E. coli is augmented and the proliferation is prominent in the ileum of CD and rectum and sigmoid of both UC and CD patients which are sites where the lesions usually are observed. The augmented E. coli population in these sites presented a low number of the virulence markers, possibly meaning that they are not relevant for the disease process.

Presence of a Human Diarrheagenic Escherichia coli Clone in Captivity Kept Psittacidaes.

Bacterial cultures of cloaca swabs from 86 captivity kept psittacidaes revealed 17 Escherichia coli bearing birds sharing strains which, on the basis of enterobacterial repetitive intergenic consensus (ERIC) PCR analysis, proved to be genetically similar. Further, triplex PCR specific for the genetic markers chuA, yjaA, and TSPE4.C2 was used to assign the strains to the E. coli reference collection (EcoR) B2 group. One strain of each, from the enteropathogenic (EPEC), enteroaggregative (EAEC) and Shiga toxin (STEC) E. coli pathovars were found among these isolates.

High prevalence of aggregative adherent Escherichia coli strains in the mucosa-associated microbiota of patients with inflammatory bowel diseases.

The intestinal population of Escherichia coli is increased in patients with inflammatory bowel disease (IBD), but the reason for this elevation, the particular features of these bacteria and their potential role in the pathogenesis of the disease are not known. The present study was undertaken to investigate the adherence abilities and some virulence properties of a collection of 131 E. coli isolates cultured from rectal biopsies of 23 subjects diagnosed with ulcerative colitis (UC), 8 with Crohn's disease (CD) and 23 control patients from southern Brazil. The adherence abilities of the bacteria were investigated in vitro, using HEp-2 epithelial cells in assays of 3 and 6h of bacteria-cell contact. The isolates were screened by PCR with primers for the following virulence genetic markers: plasmid of aggregative adhesion (pAA) and the aggregative adherence fimbriae R (aggR), E. coli attaching and effacing (eae), invasion-associated locus (ial), invasion plasmid antigen H (ipaH) and Shiga citotoxin-encoding (stx) genes. HEp-2 cells aggregative adherent E. coli strains, as detected in the 3h adherence assay, were found in 14/23 (60.9%) patients with UC, 7/8 (87.5%) with CD and in 7/23 (30.4%) controls (p=0.011). Virulence genetic markers were detected in strains of 9 patients with UC (39.1%), but in none of CD or control group. Two of these UC patients had strains harboring both pAA and aggR, one had strains positive for aggR, four had strains positive for eae and two had strains positive for stx. These results suggest that the augmented population of E. coli on the rectal mucosa of IBD patients, particularly of those diagnosed with UC, is mostly comprised of aggregative adherent strains, some of which possessing classical virulence markers of E. coli.

Reduced etiological role for enteropathogenic Escherichia coli in cases of diarrhea in Brazilian infants.

Previously common in Brazil, enteropathogenic Escherichia coli (EPEC) strains of serogroups O55, O111, and O119 are now rare, while enteroadherent strains other than EPEC, belonging to serogroups such as O125, were prevalent among 126 diarrheic infants less than 1 year old who were surveyed. None of these strains had the EPEC bundle-forming pilus (bfpA) gene.

Niche-specific association of Aeromonas Ribotypes from human and environmental origin.

A total of 88 Aeromonas isolates from distinct locations and sources (39 from extraintestinal infections, 31 from diarrhoeic, ten from non-diarrhoeic faeces, all human, and eight from fresh water) were subjected to phenospecies identification, serotyping, ribotyping and detection of some virulence markers. The strains belonged to four different phenospecies marked by 19 O serogroups and 38 ribotypes. No strong correlation between these parameters was found, and no group, as defined by the typing methods, could be characterized with a particular set of virulence markers. There was a clear association of ribotypes with the source of the strains. Cluster analysis allowed the identification of a complex of ribotypes belonging to distinct but related sources, including clinical and environmental isolates. These results suggest that ribotyping may be an epidemiological tool suitable for the study of Aeromonas infections.