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.

Implication of sortase-dependent proteins of Streptococcus thermophilus in adhesion to human intestinal epithelial cell lines and bile salt tolerance.

Streptococcus thermophilus (ST) is a lactic acid bacterium widely used in dairy industry and displays several properties which could be beneficial for host. The objective of this study was to investigate, in vitro, the implication of sortase A (SrtA) and sortase-dependent proteins (SDPs) in the adhesion of ST LMD-9 strain to intestinal epithelial cells (IECs) and resistance to bile salt mixture (BSM; taurocholoate, deoxycholate, and cholate). The effect of mutations in prtS (protease), mucBP (MUCin-Binding Protein), and srtA genes in ST LMD-9 in these mechanisms were examined. The HT29-MTX, HT29-CL.16E, and Caco-2 TC7 cell lines were used. HT29-MTX and HT29-CL.16E cells express different mucins found in the gastro intestinal tract; whereas, Caco-2 TC7 express cell surface proteins found in the small intestine. All mutants showed different adhesion profiles depending on cell lines. The mutation in genes srtA and mucBP leads to a significant decrease in LMD-9 adhesion capacity to Caco-2 TC7 cells. A mutation in mucBP gene has also shown a significant decrease in LMD-9 adhesion capacity to HT29-CL.16E cells. However, no difference was observed using HT29-MTX cells. Furthermore, ST LMD-9 and srtA mutant were resistant to BSM up to 3 mM. Contrariwise, no viable bacteria were detected for prtS and mucBP mutants at this concentration. Two conclusions could be drawn. First, SDPs could be involved in the LMD-9 adhesion depending on the cell lines indicating the importance of eukaryotic-cell surface components in adherence. Second, SDPs could contribute to resistance to bile salts probably by maintaining the cell membrane integrity.

Use of the dynamic gastro-intestinal model TIM to explore the survival of the yogurt bacterium Streptococcus thermophilus and the metabolic activities induced in the simulated human gut.

Streptococcus thermophilus, a lactic acid bacterium used to produce yogurts and cheeses is more and more considered for its potential probiotic properties. This implies that additional information should be obtained regarding its survival and metabolic activity in the human Gastro-Intestinal Tract (GIT). In this study, we screened 30 S. thermophilus strains for urease, small heat shock protein, and amino-acid decarboxylase functions which may play a role in survival in the upper part of the GIT. The survival kinetics of 4 strains was investigated using the TIM, a physiologically relevant in vitro dynamic gastric and small intestinal model. The three strains LMD9, PB18O and EBLST20 showed significantly higher survival than CNRZ21 in all digestive compartments of the TIM, which may be related to the presence of urease and heat shock protein functions. When LMD9 bacterial cells were delivered in a fermented milk formula, a significant improvement of survival in the TIM was observed compared to non-fermented milk. With the RIVET (Recombinase In Vivo Expression Technology) method applied to the LMD9 strain, a promoter located upstream of hisS, responsible for the histidyl-transfer RNA synthesis, was found to be specifically activated in the artificial stomach. The data generated on S. thermophilus survival and its adaptation capacities to the digestive tract are essential to establish a list of biomarkers useful for the selection of probiotic strains.

Runoff microbiome quality assessment of a city center rainwater harvesting zone shows a differentiation of pathogen loads according to human mobility patterns.

The hygienic quality of urban surfaces can be impaired by multiple sources of microbiological contaminants. These surfaces can trigger the development of multiple bacterial taxa and favor their spread during rain events through the circulation of runoff waters. These runoff waters are commonly directed toward sewer networks, stormwater infiltration systems or detention tanks prior a release into natural water ways. With water scarcity becoming a major worldwide issue, these runoffs are representing an alternative supply for some usage like street cleaning and plant watering. Microbiological hazards associated with these urban runoffs, and surveillance guidelines must be defined to favor these uses. Runoff microbiological quality from a recently implemented city center rainwater harvesting zone was evaluated through classical fecal indicator bacteria (FIB) assays, quantitative PCR and DNA meta-barcoding analyses. The incidence of socio-urbanistic patterns on the organization of these urban microbiomes were investigated. FIB and DNA from Human-specific Bacteroidales and pathogens such as Staphylococcus aureus were detected from most runoffs and showed broad distribution patterns. 16S rRNA DNA meta-barcoding profilings further identified core recurrent taxa of health concerns like Acinetobacter, Mycobacterium, Aeromonas and Pseudomonas, and divided these communities according to two main groups of socio-urbanistic patterns. One of these was highly impacted by heavy traffic, and showed recurrent correlation networks involving bacterial hydrocarbon degraders harboring significant virulence properties. The tpm-based meta-barcoding approach identified some of these taxa at the species level for more than 30 genera. Among these, recurrent pathogens were recorded such as P. aeruginosa, P. paraeruginosa, and Aeromonas caviae. P. aeruginosa and A. caviae tpm reads were found evenly distributed over the study site but those of P. paraeruginosa were higher among sub-catchments impacted by heavy traffic. Health risks associated with these runoff P. paraeruginosa emerging pathogens were high and associated with strong cytotoxicity on A549 lung cells. Recurrent detections of pathogens in runoff waters highlight the need of a microbiological surveillance prior allowing their use. Good microbiological quality can be obtained for certain typologies of sub-catchments with good hygienic practices but not all. A reorganization of Human mobility and behaviors would likely trigger changes in these bacterial diversity patterns and reduce the occurrences of the most hazardous groups.

Relative Weight of Organic Waste Origin on Compost and Digestate 16S rRNA Gene Bacterial Profilings and Related Functional Inferences.

Even though organic waste (OW) recycling via anaerobic digestion (AD) and composting are increasingly used, little is known about the impact of OW origin (fecal matters and food and vegetable wastes) on the end products' bacterial contents. The hypothesis of a predictable bacterial community structure in the end products according to the OW origin was tested. Nine OW treatment plants were selected to assess the genetic structure of bacterial communities found in raw OW according to their content in agricultural and urban wastes and to estimate their modifications through AD and composting. Two main bacterial community structures among raw OWs were observed and matched a differentiation according to the occurrences of urban chemical pollutants. Composting led to similar 16S rRNA gene OTU profiles whatever the OW origin. With a significant shift of about 140 genera (representing 50% of the bacteria), composting was confirmed to largely shape bacterial communities toward similar structures. The enriched taxa were found to be involved in detoxification and bioremediation activities. This process was found to be highly selective and favorable for bacterial specialists. Digestates showed that OTU profiles differentiated into two groups according to their relative content in agricultural (manure) and urban wastes (mainly activated sludge). About one third of the bacterial taxa was significantly affected by AD. In digestates of urban OW, this sorting led to an enrichment of 32 out of the 50 impacted genera, while for those produced from agricultural or mixed urban/agricultural OW (called central OW), a decay of 54 genera over 60 was observed. Bacteria from activated sludge appeared more fit for AD than those of other origins. Functional inferences showed AD enriched genera from all origins to share similar functional traits, e.g., chemoheterotrophy and fermentation, while being often taxonomically distinct. The main functional traits among the dominant genera in activated sludge supported a role in AD. Raw OW content in activated sludge was found to be a critical factor for predicting digestate bacterial contents. Composting generated highly predictable and specialized community patterns whatever the OW origin. AD and composting bacterial changes were driven by functional traits selected by physicochemical factors such as temperature and chemical pollutants.

Identification of Streptococcus thermophilus Genes Specifically Expressed under Simulated Human Digestive Conditions Using R-IVET Technology.

Despite promising health effects, the probiotic status of Streptococcus thermophilus, a lactic acid bacterium widely used in dairy industry, requires further documentation of its physiological status during human gastrointestinal passage. This study aimed to apply recombinant-based in vivo technology (R-IVET) to identify genes triggered in a S. thermophilus LMD-9 reference strain under simulated digestive conditions. First, the R-IVET chromosomal cassette and plasmid genomic library were designed to positively select activated genes. Second, recombinant clones were introduced into complementary models mimicking the human gut, the Netherlands Organization for Applied Scientific Research (TNO) gastrointestinal model imitating the human stomach and small intestine, the Caco-2 TC7 cell line as a model of intestinal epithelium, and anaerobic batch cultures of human feces as a colon model. All inserts of activated clones displayed a promoter activity that differed from one digestive condition to another. Our results also showed that S. thermophilus adapted its metabolism to stressful conditions found in the gastric and colonic competitive environment and modified its surface proteins during adhesion to Caco-2 TC7 cells. Activated genes were investigated in a collection of S. thermophilus strains showing various resistance levels to gastrointestinal stresses, a first stage in the identification of gut resistance markers and a key step in probiotic selection.

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.

Spatio-temporal variations in chemical pollutants found among urban deposits match changes in thiopurine S-methyltransferase-harboring bacteria tracked by the tpm metabarcoding approach.

The bTPMT (bacterial thiopurine S-methyltransferase), encoded by the tpm gene, can detoxify metalloid-containing oxyanions and xenobiotics. The hypothesis of significant relationships between tpm distribution patterns and chemical pollutants found in urban deposits was investigated. The tpm gene was found conserved among eight bacterial phyla with no sign of horizontal gene transfers but a predominance among gammaproteobacteria. A DNA metabarcoding approach was designed for tracking tpm-harboring bacteria among polluted urban deposits and sediments recovered for more than six years in a detention basin (DB). This DB recovers runoff waters and sediments from a zone of high commercial activities. The PCR products from DB samples led to more than 540,000 tpm reads after DADA2 or MOTHUR bio-informatic manipulations that were allocated to more than 88 and less than 634 sequence variants per sample. The tpm community patterns were significantly different between the recent urban deposits and those that had accumulated for more than 2 years in the DB, and between those of the DB surface and the DB settling pit. These groups of samples had distinct mixture of priority pollutants. Significant relationships between tpm ordination patterns, sediment accumulation time periods and location, and concentrations in PAH, chlorpyrifos, and 4-nonylphenols (NP) were observed. These correlations matched the higher occurrences of, among others, Aeromonas, Pseudomonas, and Xanthomonas tpm-harboring bacteria in recent urban DB deposits more contaminated with chrysene and alkylphenol ethoxylates. Highly significant drops in tpm reads allocated to Aeromonas species were recorded in the oldest DB sediments accumulating naphthalene and metallic pollutants. Degraders of urban pollutants such as P. aeruginosa and P. putida showed conserved distribution patterns over time but P. syringae phytopathogens were more abundant in the oldest sediments. TPMT-harboring bacteria can be used to assess the incidence of high risk priority pollutants on environmental systems.

Milk Fat Globules Hamper Adhesion of Enterohemorrhagic Escherichia coli to Enterocytes: In Vitro and in Vivo Evidence.

Enterohemorrhagic Escherichia coli (EHEC; E. coli) are food-borne agents associated with gastroenteritis, enterocolitis, bloody diarrhea and the hemolytic-uremic syndrome (HUS). Bovine milk glycans have been shown to contain oligosaccharides which are similar to host epithelial cell receptors and can therefore prevent bacterial adhesion. This study aimed to describe interactions between EHEC O157:H7 EDL933 and O26:H11 21765 and milk fat globules (MFGs) in raw milk and raw milk cheese, and the impact of MFGs on EHEC strains adhesion to the intestinal tract in vitro and in vivo. Both EHEC serotypes clearly associated with native bovine MFGs and significantly limited their adhesion to a co-culture of intestinal cells. The presence of MFGs in raw milk cheese had two effects on the adhesion of both EHEC serotypes to the intestinal tracts of streptomycin-treated mice. First, it delayed and reduced EHEC excretion in mouse feces for both strains. Second, the prime implantation site for both EHEC strains was 6 cm more proximal in the intestinal tracts of mice fed with contaminated cheese containing less than 5% of fat than in those fed with contaminated cheese containing 40% of fat. Feeding mice with 40% fat cheese reduced the intestinal surface contaminated with EHEC and may therefore decrease severity of illness.

New Insights into the Proteolytic System of Streptococcus thermophilus: Use of Isracidin To Characterize Cell-Associated Extracellular Peptidase Activities.

The influence on the hydrolysis of isracidin of cell-associated extracellular aminopeptidase and X-prolyl dipeptidyl peptidase activities in addition to protease PrtS of Streptococcus thermophilus strains was investigated. S. thermophilus LMD-9 (PrtS(+) phenotype) efficiently hydrolyzed the isracidin mainly through the PrtS activity, whereas strain CNRZ1066 (PrtS(-) phenotype) and two mutant strains LMD-9-ΔprtS and LMD-9-ΔprtS-ΔhtrA also displayed substrate hydrolysis, but different from that of the wild type strain LMD-9. Identification by mass spectrometry of breakdown products of isracidin revealed the existence of novel cell-associated extracellular carboxypeptidase and peptidyl dipeptidase activities in all PrtS(-) strains, besides known cell-associated extracellular aminopeptidase and X-prolyl dipeptidyl peptidase activities. Both aminopeptidase and peptidyl dipeptidase activities were not able to cleave the isracidin at peptide bonds with proline residues. No hydrolysis of isracidin was detected in cell free filtrate for all the strains studied, indicating that no cell lysis had occurred. Taken together, these results suggested the presence of cell-associated extracellular peptidase activities in S. thermophilus strains that could be vital for the growth of PrtS(-) strains.

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).