Spatial and temporal modulation of enterotoxigenic E. coli H10407 pathogenesis and interplay with microbiota in human gut models.

BACKGROUND: Enterotoxigenic Escherichia coli (ETEC) substantially contributes to the burden of diarrheal illnesses in developing countries. With the use of complementary in vitro models of the human digestive environment, TNO gastrointestinal model (TIM-1), and Mucosal Simulator of the Human Intestinal Microbial Ecosystem (M-SHIME), we provided the first detailed report on the spatial-temporal modulation of ETEC H10407 survival, virulence, and its interplay with gut microbiota. These systems integrate the main physicochemical parameters of the human upper digestion (TIM-1) and simulate the ileum vs ascending colon microbial communities and luminal vs mucosal microenvironments, captured from six fecal donors (M-SHIME). RESULTS: A loss of ETEC viability was noticed upon gastric digestion, while a growth renewal was found at the end of jejunal and ileal digestion. The remarkable ETEC mucosal attachment helped to maintain luminal concentrations above 6 log10 mL-1 in the ileum and ascending colon up to 5 days post-infection. Seven ETEC virulence genes were monitored. Most of them were switched on in the stomach and switched off in the TIM-1 ileal effluents and in a late post-infectious stage in the M-SHIME ascending colon. No heat-labile enterotoxin production was measured in the stomach in contrast to the ileum and ascending colon. Using 16S rRNA gene-based amplicon sequencing, ETEC infection modulated the microbial community structure of the ileum mucus and ascending colon lumen. CONCLUSIONS: This study provides a better understanding of the interplay between ETEC and gastrointestinal cues and may serve to complete knowledge on ETEC pathogenesis and inspire novel prophylactic strategies for diarrheal diseases.

Strand-specific transcriptomes of Enterohemorrhagic Escherichia coli in response to interactions with ground beef microbiota: interactions between microorganisms in raw meat.

BACKGROUND: Enterohemorrhagic Escherichia coli (EHEC) are zoonotic agents associated with outbreaks worldwide. Growth of EHEC strains in ground beef could be inhibited by background microbiota that is present initially at levels greater than that of the pathogen E. coli. However, how the microbiota outcompetes the pathogenic bacteria is unknown. Our objective was to identify metabolic pathways of EHEC that were altered by natural microbiota in order to improve our understanding of the mechanisms controlling the growth and survival of EHECs in ground beef. RESULTS: Based on 16S metagenomics analysis, we identified the microbial community structure in our beef samples which was an essential preliminary for subtractively analyzing the gene expression of the EHEC strains. Then, we applied strand-specific RNA-seq to investigate the effects of this microbiota on the global gene expression of EHEC O2621765 and O157EDL933 strains by comparison with their behavior in beef meat without microbiota. In strain O2621765, the expression of genes connected with nitrate metabolism and nitrite detoxification, DNA repair, iron and nickel acquisition and carbohydrate metabolism, and numerous genes involved in amino acid metabolism were down-regulated. Further, the observed repression of ftsL and murF, involved respectively in building the cytokinetic ring apparatus and in synthesizing the cytoplasmic precursor of cell wall peptidoglycan, might help to explain the microbiota's inhibitory effect on EHECs. For strain O157EDL933, the induced expression of the genes implicated in detoxification and the general stress response and the repressed expression of the peR gene, a gene negatively associated with the virulence phenotype, might be linked to the survival and virulence of O157:H7 in ground beef with microbiota. CONCLUSION: In the present study, we show how RNA-Seq coupled with a 16S metagenomics analysis can be used to identify the effects of a complex microbial community on relevant functions of an individual microbe within it. These findings add to our understanding of the behavior of EHECs in ground beef. By measuring transcriptional responses of EHEC, we could identify putative targets which may be useful to develop new strategies to limit their shedding in ground meat thus reducing the risk of human illnesses.

Increased EHEC survival and virulence gene expression indicate an enhanced pathogenicity upon simulated pediatric gastrointestinal conditions.

BACKGROUND: Enterohemorrhagic Escherichia coli (EHEC) are major foodborne pathogens that constitute a serious public health threat, mainly in young children. Shiga toxins (Stx) are the main virulence determinants of EHEC pathogenesis but adhesins like intimin (eae) and Long polar fimbriae (Lpf) also contribute to infection. The TNO GastroIntestinal Model (TIM) was used for a comparative study of EHEC O157:H7 survival and virulence under adult and child digestive conditions. METHODS: Survival kinetics in the in vitro digestive tract were determined by plating while bacterial viability was assessed by flow cytometry analysis. Expression of stx, eae, and lpf genes was followed by reverse transcriptase-quantitative PCR (RT-qPCR) and Stx production was measured by ELISA (enzyme-linked immunosorbent assay). RESULTS: Upon gastrointestinal passage, a higher amount of viable cells was found in the simulated ileal effluents of children compared to that of adults (with 34 and 6% of viable cells, respectively). Expression levels of virulence genes were up to 125-fold higher in children. Stx was detected only in child ileal effluents. CONCLUSION: Differences in digestive physicochemical parameters may partially explain why children are more susceptible to EHEC infection than adults. Such data are essential for a full understanding of EHEC pathogenesis and would help in designing novel therapeutic approaches.

Occurrence of 40 sanitary indicators in French digestates derived from different anaerobic digestion processes and raw organic wastes from agricultural and urban origin.

This study investigated the sanitary quality of digestates resulting from the mesophilic anaerobic digestion (AD) of urban and agricultural organic wastes (OWs). 40 sanitary indicators, including pathogenic bacteria, antimicrobial resistance genes, virulence factor genes, and mobile genetic elements were evaluated using real-time PCR and/or droplet digital PCR. 13 polycyclic aromatic hydrocarbons (PAHs) and 13 pharmaceutical products (PHPs) were also measured. We assessed agricultural OWs from three treatment plants to study the effect of different AD processes (feeding mode, number of stages, pH), and used three laboratory-scale reactors to study the effect of different feed-supplies (inputs). The lab-scale reactors included: Lab1 fed with 97% activated sludge (urban waste) and 3% cow manure; Lab2 fed with 85% sludge-manure mixture supplemented with 15% wheat straw (WS); and Lab3 fed with 81% sludge-manure mixture, 15% WS, and 4% zeolite powder. Activated sludge favored the survival of the food-borne pathogens Clostridium perfringens and Bacillus cereus, carrying the toxin-encoding genes cpe and ces, respectively. Globally, the reactors fed with fecal matter supplemented with straw (Lab2) or with straw and zeolite (Lab3) had a higher hygienization efficiency than the reactor fed uniquely with fecal matter (Lab1). Three pathogenic bacteria (Enterococcus faecalis, Enterococcus faecium, and Mycobacterium tuberculosis complex), a beta-lactam resistance gene (bla TEM), and three mobile genetic elements (intI1, intI2, and IS26) were significantly decreased in Lab2 and Lab3. Moreover, the concentrations of 11 PAHs and 11 PHPs were significantly lower in Lab2 and Lab3 samples than in Lab1 samples. The high concentrations of micropollutants, such as triclosan, found in Lab1, could explain the lower hygienization efficiency of this reactor. Furthermore, the batch-fed reactor had a more efficient hygienization effect than the semi-continuous reactors, with complete removal of the ybtA gene, which is involved in the production of the siderophore yersiniabactin, and significant reduction of intI2 and tetO. These data suggest that it is essential to control the level of chemical pollutants in raw OWs to optimize the sanitary quality of digestates, and that adding co-substrate, such as WS, may overcome the harmful effect of pollutants.

Multi-targeted properties of the probiotic saccharomyces cerevisiae CNCM I-3856 against enterotoxigenic escherichia coli (ETEC) H10407 pathogenesis across human gut models.

Enterotoxigenic Escherichia coli (ETEC) is one of the most common causes of acute traveler's diarrhea. Adhesins and enterotoxins constitute the major ETEC virulence traits. With the dramatic increase in antibiotic resistance, probiotics are considered a wholesome alternative to prevent or treat ETEC infections. Here, we examined the antimicrobial properties of the probiotic Saccharomyces cerevisiae CNCM I-3856 against ETEC H10407 pathogenesis upon co-administration in the TNO gastrointestinal Model (TIM-1), simulating the physicochemical and enzymatic conditions of the human upper digestive tract and preventive treatment in the Mucosal Simulator of the Human Intestinal Microbial Ecosystem (M-SHIME), integrating microbial populations of the ileum and ascending colon. Interindividual variability was assessed by separate M-SHIME experiments with microbiota from six human individuals. The probiotic did not affect ETEC survival along the digestive tract. However, ETEC pathogenicity was significantly reduced: enterotoxin encoding virulence genes were repressed, especially in the TIM-1 system, and a lower enterotoxin production was noted. M-SHIME experiments revealed that 18-days probiotic treatment stimulate the growth of Bifidobacterium and Lactobacillus in different gut regions (mucosal and luminal, ileum and ascending colon) while a stronger metabolic activity was noted in terms of short-chain fatty acids (acetate, propionate, and butyrate) and ethanol production. Moreover, the probiotic pre-treated microbiota displayed a higher robustness in composition following ETEC challenge compared to the control condition. We thus demonstrated the multi-inhibitory properties of the probiotic S. cerevisiae CNCM I-3856 against ETEC in the overall simulated human digestive tract, regardless of the inherent variability across individuals in the M-SHIME.

The Effect of Mixing Milk of Different Species on Chemical, Physicochemical, and Sensory Features of Cheeses: A Review.

The yield and quality of cheese are associated with the composition, physicochemical, sensory, rheological, and microbiological properties of milk and with the technology applied to the milk before and/or during cheese processing. This review describes the most important research on cheeses obtained from processing mixtures of different milk species and discusses the effect of milk mixtures (i.e., species and mixture ratios) on composition, physicochemical, sensory, rheological, and microbiological properties of cheeses. More specifically, the present review paper will gather and focus only on studies that have provided a clear comparison between cheeses produced from a mixture of two milk species to cheeses produced from only one species.

Biochemical, Physicochemical and Sensory Properties of Yoghurts Made from Mixing Milks of Different Mammalian Species.

Among developed countries, bovine milk production makes a major contribution towards the economy. Elevating consumer demand for functional foods has triggered a niche for non-bovine milk-based products. Mixing milks from different species can be a strategy to increase the consumption of non-bovine milk and enable consumers and dairy companies to benefit from their nutritional and technological advantages. Thus, this review aimed to gather the most important research on yoghurts derived from processing mixtures of milks of different species. We discuss the impact of milk mixtures (i.e., species and milk ratio) on nutritional, physicochemical, sensory, rheological and microbiological properties of yoghurts. More specifically, this paper only highlights studies that have provided a clear comparison between yoghurts processed from a mixture of two milk species and yoghurts processed from a single species of milk. Finally, certain limitations and future trends are discussed, and some recommendations are suggested for future research.

2D-Cross Correlation Spectroscopy Coupled with Molecular Fluorescence Spectroscopy for Analysis of Molecular Structure Modification of Camel Milk and Cow Milk Mixtures during Coagulation.

Synchronous fluorescence spectroscopy (SFS) coupled with two-dimensional correlation spectroscopy (2DCOS) was employed to monitor, at the molecular level, the coagulation of five mixture ratios of camel's milk (CaM) and cow's milk (CM) (100% CaM, 75% CaM:25% CM, 50% CaM:50% CM, 25% CaM:75% CM and 100% CM). The dissimilarities among the different formulations are highlighted on the synchronous 2DCOS-SFS. In addition, according to the cross-peak symbols in synchronous and asynchronous spectra, the rate of response modification in riboflavin, protein and vitamin A matched with common coagulation phenomena usually reported during chymosin coagulation (hydrolysis of κ-casein, destabilization of casein micelles and aggregation). This study demonstrated that 2DCOS-SFS is a successful strategy to discriminate milk mixtures and to monitor molecular structure modifications during coagulation process.

Fluorescence spectroscopy as a promising tool for a polyphasic approach to pseudomonad taxonomy.

Fluorescence spectroscopy is an emerging tool for the analysis of biomolecules from complex matrices. We explored the potentialities of the method for the pseudomonad taxonomic purpose at the genus and species level. Emission spectra of three intrinsic fluorophores (namely, NADH, tryptophan, and the complex of aromatic amino acids and nucleic acid) were collected from whole bacterial cells. Their comparisons were performed through principal component analysis and factorial discriminant analysis. Reference strains from the Xanthomonas, Stenotrophomonas, Burkholderia, and Pseudomonas genera were well separated, with sensitivity and selectivity higher than 90%. At the species level, P. lundensis, P. taetrolens, P. fragi, P. chlororaphis, and P. stutzeri were also well separated, in a distant group, from P. putida, P. pseudoalcaligenes, and P. fluorescens. These results are in agreement with the generally admitted rRNA and DNA bacterial homology grouping but they also provide additional information about strain relatedness. In the case of environmental isolates, the method allows good discrimination, even for strains for which ambiguity still remained after PCR and API 20NE identification. Rapid, easy to perform, and low cost, fluorescence spectroscopy provides substantial information on cell components. Statistical analysis of collected data allows in-depth comparison of strains. Our results strongly support the view that fluorescence spectroscopy fingerprinting can be used as a powerful tool in a polyphasic approach to pseudomonad taxonomy.

Genome Sequence and Annotation of a Human Infection Isolate of Escherichia coli O26:H11 Involved in a Raw Milk Cheese Outbreak.

The consumption of raw milk cheese can expose populations to Shiga toxin-producing Escherichia coli (STEC). We report here the genome sequence of an E. coli O26:H11 strain isolated from humans during the first raw milk cheese outbreak described in France (2005).

Alteration of raw-milk cheese by Pseudomonas spp.: monitoring the sources of contamination using fluorescence spectroscopy and metabolic profiling.

Thirty Pseudomonas spp. strains isolated from milk, water, cheese centre and cheese surface in two traditional workshops manufacturing raw milk St. Nectaire cheese were characterised by fluorescence spectroscopy and Biolog metabolic profiling. Factorial discriminant analysis (FDA) of the two data sets revealed clear linkages between groups of isolates. In the first workshop, milk could be incriminated as the sole source of cheese contamination. In the second one, milk and cheese centre isolates were found similar but surface cheese contaminants could be linked to a secondary contamination originating from water. Thus, it is possible to characterise, differentiate and trace Pseudomonas spp. strains using the fluorescence and metabolic profiling techniques. In addition, the two data sets were found highly correlated by canonical correlation analysis (CCA). Fluorescence spectroscopy however showed several advantages because of its low cost and processing speed.