Commensal Escherichia coli Strains of Bovine Origin Competitively Mitigated Escherichia coli O157:H7 in a Gnotobiotic Murine Intestinal Colonization Model with or without Physiological Stress.

Cattle are a primary reservoir of enterohemorrhagic Escherichia coli (EHEC) O157:H7. Currently, there are no effective methods of eliminating this important zoonotic pathogen from cattle, and colonization resistance in relation to EHEC O157:H7 in cattle is poorly understood. We developed a gnotobiotic EHEC O157:H7 murine model to examine aspects of the cattle pathogen-microbiota interaction, and to investigate competitive suppression of EHEC O157:H7 by 18 phylogenetically distinct commensal E. coli strains of bovine origin. As stress has been suggested to influence enteric colonization by EHEC O157:H7 in cattle, corticosterone administration (±) to incite a physiological stress response was included as an experimental variable. Colonization of the intestinal tract (IT) of mice by the bovine EHEC O157:H7 strain, FRIK-2001, mimicked characteristics of bovine IT colonization. In this regard, FRIK-2001 successfully colonized the IT and temporally incited minimal impacts on the host relative to other EHEC O157:H7 strains, including on the renal metabolome. The presence of the commensal E. coli strains decreased EHEC O157:H7 densities in the cecum, proximal colon, and distal colon. Moreover, histopathologic changes and inflammation markers were reduced in the distal colon of mice inoculated with commensal E. coli strains (both propagated separately and communally). Although stress induction affected the behavior of mice, it did not influence EHEC O157:H7 densities or disease. These findings support the use of a gnotobiotic murine model of enteric bovine EHEC O157:H7 colonization to better understand pathogen-host-microbiota interactions toward the development of effective on-farm mitigations for EHEC O157:H7 in cattle, including the identification of bacteria capable of competitively colonizing the IT.

Impact of ß2-1 fructan on faecal community change: results from a placebo-controlled, randomised, double-blinded, cross-over study in healthy adults.

Healthy adults (n 30) participated in a placebo-controlled, randomised, double-blinded, cross-over study consisting of two 28 d treatments (ß2-1 fructan or maltodextrin; 3×5 g/d) separated by a 14-d washout. Subjects provided 1 d faecal collections at days 0 and 28 of each treatment. The ability of faecal bacteria to metabolise ß2-1 fructan was common; eighty-seven species (thirty genera, and four phyla) were isolated using anaerobic medium containing ß2-1 fructan as the sole carbohydrate source. ß2-1 fructan altered the faecal community as determined through analysis of terminal restriction fragment length polymorphisms and 16S rRNA genes. Supplementation with ß2-1 fructan reduced faecal community richness, and two patterns of community change were observed. In most subjects, ß2-1 fructan reduced the content of phylotypes aligning within the Bacteroides, whereas increasing those aligning within bifidobacteria, Faecalibacterium and the family Lachnospiraceae. In the remaining subjects, supplementation increased the abundance of Bacteroidetes and to a lesser extent bifidobacteria, accompanied by decreases within the Faecalibacterium and family Lachnospiraceae. ß2-1 Fructan had no impact on the metagenome or glycoside hydrolase profiles in faeces from four subjects. Few relationships were found between the faecal bacterial community and various host parameters; Bacteroidetes content correlated with faecal propionate, subjects whose faecal community contained higher Bacteroidetes produced more caproic acid independent of treatment, and subjects having lower faecal Bacteroidetes exhibited increased concentrations of serum lipopolysaccharide and lipopolysaccharide binding protein independent of treatment. We found no evidence to support a defined health benefit for the use of ß2-1 fructans in healthy subjects.

Antimicrobial growth promoter use in livestock: a requirement to understand their modes of action to develop effective alternatives.

Antimicrobial agents (AMAs) have been used in agriculture since the 1950s as growth-promoting agents [antimicrobial growth promoters (AGPs)]. They have provided benefits to the agricultural industry by increasing production efficiencies and maximising livestock health, yet the potential risks surrounding resistance to AMAs in medically important pathogenic bacteria have enhanced public and government scrutiny regarding AMA use in agriculture. Although it is recognised that AGP administration can select for resistance to AMAs in enteric bacteria of livestock, conclusive evidence showing a link between resistant bacteria from livestock and human health is lacking (e.g. transmission of resistant zoonotic pathogens). Livestock production output must be increased significantly due to the increase in global population, and thus the identification of non-AMA alternatives to AGP use is required. One strategy employed to identify alternatives to AGPs is an observational empirical methodology, but this approach has failed to deliver effective alternatives. A second approach is aimed at understanding the mechanisms involved in AGP function and developing alternatives that mimic the physiological responses to AGPs. New evidence indicates that AGP function is more complex than merely affecting enteric bacterial populations, and AGPs likely function by directly or indirectly modulating host responses such as the immune system. As such, a more comprehensive understanding of the mechanisms associated with AMA function as AGPs will facilitate the development of effective alternatives.

Housing influences tissue cytokine levels and the fecal bacterial community structure in rats.

Immune measures and the fecal bacterial community were examined in female Biobreeding rats housed in wire bottom cages (wire) or in solid bottom cages containing hardwood chips (bedding). Housing did not affect food intake, weight gain, fecal output or fibre content, serum liver enzymes, or spleen and mesenteric lymph node immune cell populations. Bedding-housed rat feces were enriched in phylotypes aligning within the phylum Firmicutes (families Lactobacillaceae and Erysipelotrichaceae) and had a 2-fold lower content of phylotypes aligning within the phylum Bacteroidetes. Feces from bedding-housed rats also contained significantly more acetic acid and less propionic, isobutyric, valeric and isovaleric acids than those housed on wire. Bedding-housed rats had significantly higher splenic concentrations of interleukin-4 (P < 0.001). These results demonstrate that bedding can indirectly influence systemic and mucosal immune measures, potentially adding additional complexities and confounding results to nutrition studies investigating the health effects of dietary fibres.

Modeling the behavior of Listeria monocytogenes during enrichment in half Fraser broth; impact of pooling and the duration of enrichment on the detection of L. monocytogenes in food.

A stochastic model describing the growth of Listeria monocytogenes during enrichment in half Fraser was developed for the purpose of estimating the effects of modifications to the first enrichment step of the EN ISO 11290-1 detection method. Information pertaining to the variability of growth rates, physiological state of the cell, and the behavior of individual cells contaminating the food were obtained from previously published studies. We used this model to investigate the impact of pooling enrichment broths (wet pooling) on the performance of the standard method. For validation of the model, the numbers of L. monocytogenes occurring in 88 naturally contaminated foods following pre-enrichment were compared to model-simulated microbial counts. The model was then used to perform simulations representative of the natural contamination observed for smoked salmon in the European baseline survey of 2010-2011. The model-estimated L. monocytogenes levels following individual enrichment or following the pooling of five broths where only one would be contaminated were compared. The model indicated a 10% loss of method sensitivity resulting from wet pooling. The model also predicted a 5% decrease in the sensitivity of the method when the duration of the enrichment was reduced from 24 to 22 h.

ß2-1 Fructan supplementation alters host immune responses in a manner consistent with increased exposure to microbial components: results from a double-blinded, randomised, cross-over study in healthy adults.

ß2-1 Fructans are purported to improve health by stimulating growth of colonic bifidobacteria, increasing host resistance to pathogens and stimulating the immune system. However, in healthy adults, the benefits of supplementation remain undefined. Adults (thirteen men, seventeen women) participated in a double-blinded, placebo-controlled, randomised, cross-over study consisting of two 28-d treatments separated by a 14-d washout period. Subjects' regular diets were supplemented with ß2-1 fructan or placebo (maltodextrin) at 3×5 g/d. Fasting blood and 1-d faecal collections were obtained at the beginning and at the end of each phase. Blood was analysed for clinical, biochemical and immunological variables. Determinations of well-being and general health, gastrointestinal (GI) symptoms, regularity, faecal SCFA content, residual faecal ß2-1 fructans and faecal bifidobacteria content were undertaken. ß2-1 Fructan supplementation had no effect on blood lipid or cholesterol concentrations or on circulating lymphocyte and macrophage numbers, but significantly increased serum lipopolysaccharide, faecal SCFA, faecal bifidobacteria and indigestion. With respect to immune function, ß2-1 fructan supplementation increased serum IL-4, circulating percentages of CD282+/TLR2+ myeloid dendritic cells and ex vivo responsiveness to a toll-like receptor 2 agonist. ß2-1 Fructans also decreased serum IL-10, but did not affect C-reactive protein or serum/faecal Ig concentrations. No differences in host well-being were associated with either treatment, although the self-reported incidence of GI symptoms and headaches increased during the ß2-1 fructan phase. Although ß2-1 fructan supplementation increased faecal bifidobacteria, this change was not directly related to any of the determined host parameters.

Evaluation of reduction of Fraser incubation by 24h in the EN ISO 11290-1 standard on detection and diversity of Listeria species.

The EN ISO 11290-1 method for the isolation of Listeria monocytogenes from food is carried out using a double enrichment in Fraser broths. While the method is effective it is also quite long requiring 4-7 days to process a contaminated food, and may be adversely affected by inter-strain and/or inter-species competition in samples containing mixed Listeria populations. Currently, we have little information on the impact of competition on food testing under routine conditions. Food samples (n=130) were analyzed using the standard method and the evolution of Listeria populations in 89 naturally contaminated samples followed over the entire enrichment process. In most instances, maximum increase in L. monocytogenes population occurred over the first 24h following sub-culture in Full Fraser broth and strain recovery was similar at both 24 and 48 h, indicating that the second enrichment step can be reduced by 24h without impacting the recovery of L. monocytogenes or affecting the sensitivity of the method. In approximately 6% of naturally contaminated samples the presence of competing Listeria species adversely impacted L. monocytogenes population levels. Moreover, these effects were more pronounced during the latter 24h of the Fraser enrichment, and potentially could affect or complicate the isolation of these strains.

A Spatially Continuous Model of Carbohydrate Digestion and Transport Processes in the Colon.

A spatially continuous mathematical model of transport processes, anaerobic digestion and microbial complexity as would be expected in the human colon is presented. The model is a system of first-order partial differential equations with context determined number of dependent variables, and stiff, non-linear source terms. Numerical simulation of the model is used to elucidate information about the colon-microbiota complex. It is found that the composition of materials on outflow of the model does not well-describe the composition of material in other model locations, and inferences using outflow data varies according to model reactor representation. Additionally, increased microbial complexity allows the total microbial community to withstand major system perturbations in diet and community structure. However, distribution of strains and functional groups within the microbial community can be modified depending on perturbation length and microbial kinetic parameters. Preliminary model extensions and potential investigative opportunities using the computational model are discussed.

Sex differences in gut fermentation and immune parameters in rats fed an oligofructose-supplemented diet.

BACKGROUND: Mechanistic data to support health claims is often generated using rodent models, and the influence of prebiotic supplementation has largely been evaluated using male rodents. Given that sex-based differences in immune parameters are well recognized and recent evidence suggests differences in microbiota composition between sexes, validation of the effectiveness of prebiotics merits assessment in both males and females. Here, we have compared the effect of oligofructose (OF) supplementation on the fecal bacterial community, short chain fatty acid profiles, and gut mucosal and systemic immune parameters in male and female rats. METHODS: Male and female rats were fed rodent chow or chow supplemented with OF (5 % w/w). Fecal community change was examined by analyzing 16S rRNA gene content. To compare effects of OF between sexes at the gut microbial and mucosal immune level, fecal short chain fatty acid and tissue cytokine profiles were measured. Serum lipopolysaccharide levels were also evaluated by the limulus amebocyte lysate assay as an indirect means of determining gut permeability between sexes. RESULTS: In the fecal community of females, OF supplementation altered community structure by increasing abundance in the Phylum Bacteroidetes. In male rats, no changes in fecal community structure were observed, although fecal butyrate levels significantly increased. Liver Immunoglobulin A (IgA) levels were higher in males relative to females fed OF, and serum LPS concentrations were higher in males independent of diet. Females had higher basal levels of the regulatory cytokine interleukin-10 (IL-10) in the colon and liver, while males had higher basal levels of the pro-inflammatory cytokines IL-6 and cytokine-induced neutrophil chemoattractant-1 (CINC-1) in the cecum and liver. CONCLUSIONS: We have shown that male and female rat gut communities metabolize an OF-supplemented diet differently. Sex-specific responses in both the fecal community and systemic immune parameters suggest that this difference may result from an increase in the availability of gut peptidyl-nitrogen in the males. These findings demonstrate the importance of performing sex-comparative studies when investigating potential health effects of prebiotics using rodent models.

Phylum level change in the cecal and fecal gut communities of rats fed diets containing different fermentable substrates supports a role for nitrogen as a factor contributing to community structure.

Fermentation differs between the proximal and distal gut but little is known regarding how the bacterial communities differ or how they are influenced by diet. In order to investigate this, we compared community diversity in the cecum and feces of rats by 16S rRNA gene content and DNA shot gun metagenomics after feeding purified diets containing different fermentable substrates. Gut community composition was dependent on the source of fermentable substrate included in the diet. Cecal communities were dominated by Firmicutes, and contained a higher abundance of Lachnospiraceae compared to feces. In feces, community structure was shifted by varying degrees depending on diet towards the Bacteroidetes, although this change was not always evident from 16S rRNA gene data. Multi-dimensional scaling analysis (PCoA) comparing cecal and fecal metagenomes grouped by location within the gut rather than by diet, suggesting that factors in addition to substrate were important for community change in the distal gut. Differentially abundant genes in each environment supported this shift away from the Firmicutes in the cecum (e.g., motility) towards the Bacteroidetes in feces (e.g., Bacteroidales transposons). We suggest that this phylum level change reflects a shift to ammonia as the primary source of nitrogen used to support continued microbial growth in the distal gut.

Phylogenetic identification of methanogens assimilating acetate-derived carbon in dairy and swine manures.

In order to develop approaches for reducing the carbon footprint of the swine and dairy industries, it is important first to identify the methanogenic communities that drive methane emissions from stored manure. In this study, the metabolically active methanogens in substrate-starved manure samples taken from two dairy and one swine manure storage tanks were identified using [(13)C]-acetate and DNA stable-isotope probing (DNA-SIP). Molecular analysis of recovered genomic [(13)C]-DNA revealed that two distinct clusters of unclassified methanogen populations affiliated with the Methanoculleus genus, and the populations affiliated with Methanoculleus chikugoensis assimilated acetate-derived carbon (acetate-C) in swine and dairy starved manure samples, respectively. Furthermore, carbon flow calculations indicated that these populations were the primary contributors to methane emissions during these anoxic SIP incubations. Comparative analysis of mcrA gene abundance (coding for a key enzyme of methanogenesis) for Methanoculleus spp. in fresh feces and a wider range of stored dairy or swine manure samples, by real-time quantitative PCR using newly designed specific primers, demonstrated that the abundance of this genus significantly increased during storage. The findings supported the involvement of these particular methanogen populations as methane emitters from swine and dairy manure storage tanks. The study revealed that the ability to assimilate acetate-C for growth in manure differed within the Methanoculleus genus.

An allelopathy based model for the Listeria overgrowth phenomenon.

In a standard procedure of food safety testing, the presence of the pathogenic bacterium Listeria monocytogenes can be masked by non-pathogenic Listeria. This phenomenon of Listeria overgrowth is not well understood. We present a mathematical model for the growth of a mixed population of L. innocua and L. monocytogenes that includes competition for a common resource and allelopathic control of L. monocytogenes by L. innocua when this resource becomes limited, which has been suggested as one potential explanation for the overgrowth phenomenon. The model is tested quantitatively and qualitatively against experimental data in batch experiments. Our results indicate that the phenomenon of masked pathogens can depend on initial numbers of each population present, and on the intensity of the allelopathic effect. Prompted by the results for the batch setup, we also analyze the model in a hypothetical chemostat setup. Our results suggest that it might be possible to operate a continuous growth environment such that the pathogens outcompete the non-pathogenic species, even in cases where they would be overgrown in a batch environment.

Spaceflight influences both mucosal and peripheral cytokine production in PTN-Tg and wild type mice.

Spaceflight is associated with several health issues including diminished immune efficiency. Effects of long-term spaceflight on selected immune parameters of wild type (Wt) and transgenic mice over-expressing pleiotrophin under the human bone-specific osteocalcin promoter (PTN-Tg) were examined using the novel Mouse Drawer System (MDS) aboard the International Space Station (ISS) over a 91 day period. Effects of this long duration flight on PTN-Tg and Wt mice were determined in comparison to ground controls and vivarium-housed PTN-Tg and Wt mice. Levels of interleukin-2 (IL-2) and transforming growth factor-beta1 (TGF-ß1) were measured in mucosal and systemic tissues of Wt and PTN-Tg mice. Colonic contents were also analyzed to assess potential effects on the gut microbiota, although no firm conclusions could be made due to constraints imposed by the MDS payload and the time of sampling. Spaceflight-associated differences were observed in colonic tissue and systemic lymph node levels of IL-2 and TGF-ß1 relative to ground controls. Total colonic TGF-ß1 levels were lower in Wt and PTN-Tg flight mice in comparison to ground controls. The Wt flight mouse had lower levels of IL-2 and TGF-ß1 compared to the Wt ground control in both the inguinal and brachial lymph nodes, however this pattern was not consistently observed in PTN-Tg mice. Vivarium-housed Wt controls had higher levels of active TGF-ß1 and IL-2 in inguinal lymph nodes relative to PTN-Tg mice. The results of this study suggest compartmentalized effects of spaceflight and on immune parameters in mice.

Promotion of autoimmune diabetes by cereal diet in the presence or absence of microbes associated with gut immune activation, regulatory imbalance, and altered cathelicidin antimicrobial Peptide.

We are exposed to millions of microbial and dietary antigens via the gastrointestinal tract, which likely play a key role in type 1 diabetes (T1D). We differentiated the effects of these two major environmental factors on gut immunity and T1D. Diabetes-prone BioBreeding (BBdp) rats were housed in specific pathogen-free (SPF) or germ-free (GF) conditions and weaned onto diabetes-promoting cereal diets or a protective low-antigen hydrolyzed casein (HC) diet, and T1D incidence was monitored. Fecal microbiota 16S rRNA genes, immune cell distribution, and gene expression in the jejunum were analyzed. T1D was highest in cereal-SPF (65%) and cereal-GF rats (53%) but inhibited and delayed in HC-fed counterparts. Nearly all HC-GF rats remained diabetes-free, whereas HC-fed SPF rats were less protected (7 vs. 29%). Bacterial communities differed in SPF rats fed cereal compared with HC. Cereal-SPF rats displayed increased gut CD3(+) and CD8α(+) lymphocytes, ratio of Ifng to Il4 mRNA, and Lck expression, indicating T-cell activation. The ratio of CD3(+) T cells expressing the Treg marker Foxp3(+) was highest in HC-GF and lowest in cereal-SPF rats. Resident CD163(+) M2 macrophages were increased in HC-protected rats. The cathelicidin antimicrobial peptide (Camp) gene was upregulated in the jejunum of HC diet-protected rats, and CAMP(+) cells colocalized with CD163. A cereal diet was a stronger promoter of T1D than gut microbes in association with impaired gut immune homeostasis.

Identification of Methanoculleus spp. as active methanogens during anoxic incubations of swine manure storage tank samples.

Methane emissions represent a major environmental concern associated with manure management in the livestock industry. A more thorough understanding of how microbial communities function in manure storage tanks is a prerequisite for mitigating methane emissions. Identifying the microorganisms that are metabolically active is an important first step. Methanogenic archaea are major contributors to methanogenesis in stored swine manure, and we investigated active methanogenic populations by DNA stable isotope probing (DNA-SIP). Following a preincubation of manure samples under anoxic conditions to induce substrate starvation, [U-(13)C]acetate was added as a labeled substrate. Fingerprint analysis of density-fractionated DNA, using length-heterogeneity analysis of PCR-amplified mcrA genes (encoding the alpha subunit of methyl coenzyme M reductase), showed that the incorporation of (13)C into DNA was detectable at in situ acetate concentrations (~7 g/liter). Fingerprints of DNA retrieved from heavy fractions of the (13)C treatment were primarily enriched in a 483-bp amplicon and, to a lesser extent, in a 481-bp amplicon. Analyses based on clone libraries of the mcrA and 16S rRNA genes revealed that both of these heavy DNA amplicons corresponded to Methanoculleus spp. Our results demonstrate that uncultivated methanogenic archaea related to Methanoculleus spp. were major contributors to acetate-C assimilation during the anoxic incubation of swine manure storage tank samples. Carbon assimilation and dissimilation rate estimations suggested that Methanoculleus spp. were also major contributors to methane emissions and that the hydrogenotrophic pathway predominated during methanogenesis.

Prebiotics and probiotics: some thoughts on demonstration of efficacy within the regulatory sphere.

Probiotics and prebiotics present regulators with challenges because they require a demonstrated positive health outcome and proof that the prebiotic or probiotic is the agent of action once safety aspects have been satisfied. Thus, probiotic and prebiotic definitions are important because they will set the criteria by which these materials will be judged within the regulatory sphere. Use of the terms probiotic and prebiotic are, themselves, considered health claims in some jurisdictions, so that both product health claims and product content labeling may be regulated. Currently accepted definitions of prebiotic and probiotic make it easier to draw a straight line between ingestion and health outcome for probiotics but much more difficult for prebiotics, where a health outcome must be linked to changes in specific bacterial species within the gut microbial community. These challenges highlight the difficulties facing regulatory bodies and the scientific community when emerging science is turned into consumable product.

Molecular methods to measure intestinal bacteria: a review.

The intestine is an exceptionally rich ecosystem encompassing a complex interaction among microorganisms, influenced by host factors, ingested food, and liquid. Characterizing the intestinal microbiota is currently an active area of research. Various molecular-based methods are available to characterize the intestinal microbiota, but all methods possess relative strengths, as well as salient weaknesses. It is important that researchers are cognizant of the limitations of these methods, and that they take the appropriate steps to mitigate weaknesses. Here, we discuss methodologies used to monitor intestinal bacteria including: (i) traditional clone libraries; (ii) direct sequencing using next-generation parallel sequencing technology; (iii) denaturing gradient gel electrophoresis and temperature gradient gel electrophoresis; (iv) terminal restriction fragment length polymorphism analysis; (v) fluorescent in situ hybridization; and (vi) quantitative PCR. In addition, we also discuss experimental design, sample collection and storage, DNA extraction, gene targets, PCR bias, and methods to reduce PCR bias.

Methanoculleus spp. as a biomarker of methanogenic activity in swine manure storage tanks.

Greenhouse gas emissions represent a major problem associated with manure management in the livestock industry. A prerequisite to mitigate methane emissions occurring during manure storage is a clearer understanding of how the microbial consortia involved in methanogenesis function. Here, we have examined manure stored in outdoor tanks from two different farms, at different locations and depths. Physico-chemical and microbiological characterization of these samples indicated differences between each tank, as well as differences within each tank dependent on the depth of sampling. The dynamics of both the bacterial and archaeal communities within these samples were monitored over a 150-day period of anaerobic incubation to identify and track emerging microorganisms, which may be temporally important in the methanogenesis process. Analyses based on DNA fingerprinting of microbial communities identified trends common among all samples as well as trends specific to certain samples. All archaeal communities became enriched with Methanoculleus spp. over time, indicating that the hydrogenotrophic pathway of methanogenesis predominated. Although the emerging species differed in samples obtained from shallow depths compared to deep samples, the temporal enrichment of Methanoculleus suggests that this genus may represent a relevant indicator of methanogenic activity in swine manure storage tanks.

A novel fingerprint method to assess the diversity of methanogens in microbial systems.

Understanding the ecology of methanogens in natural and engineered environments is a prerequisite to predicting or managing methane emissions. In this study, a novel high-throughput fingerprint method was developed for determining methanogen diversity and relative abundance within environmental samples. The method described here, designated amplicon length heterogeneity PCR of the mcrA gene (LH-mcrA), is based on the natural length variation in the mcrA gene. The mcrA gene encodes the alpha-subunit of the methyl-coenzyme M reductase, which is involved in the terminal step of methane production by methanogens. The methanogenic communities from stored swine and dairy manures were distinct from each other. To validate the method, methanogenic communities in a plug flow-type bioreactor (PFBR) treating swine manure were characterized using LH-mcrA method and correlated to mcrA gene clone libraries. The diversity and relative abundance of the methanogenic groups were assessed. Methanobrevibacter, Methanosarcinaceae, Methanoculleus, Methanogenium, Methanocorpusculum and one unidentified group were assigned to particular LH-mcrA amplicons. Particular phylotypes related to Methanoculleus were predominant in the last compartment of the PFBR where the bulk of methane was produced. LH-mcrA method was found to be a reliable, fast and cost-effective alternative for diversity assessment of methanogenic communities in microbial systems.