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.

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.

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.

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.

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.

Spatial distribution of some microbial trophic groups in a plug-flow-type anaerobic bioreactor treating swine manure.

Anaerobic digestion of swine manure is carried out by a consortium of microbial species, including volatile fatty acid (VFA) producers, VFA-degraders and methanogens. The distribution of five phylogenetic groups within a plug-flow-type anaerobic bioreactor consisting of eight serially-connected tanks was examined through the sequential digestion of swine manure. Quantification was carried out using reverse transcription real-time PCR (RT-Q-PCR) assays targeting the 16S rRNA of Clostridium (cluster XIVa), Peptostreptococcus, Syntrophomonas, Methanosaeta, and Methanosarcina spp. The VFA producers Peptostreptococcus spp. and Clostridium spp. were found predominantly in compartments where hydrolysis/acidogenesis took place. The spatial distribution of the aceticlastic methanogens, Methanosaeta and Methanosarcina, within the bioreactor was not correlated with methanogenic activity. In contrast the VFA-degrading genus Syntrophomonas spp. was more abundant in compartments with elevated methanogenic activity. Multivariate statistical analyses of the RT-Q-PCR data have provided new insights into our understanding of how the various trophic groups were distributed within this bioreactor system. While the distribution of clostridia, peptostreptococci and Syntrophomonas corresponded to their known metabolic functions, aceticlastic methanogens were not apparently linked to the methanogenesis stage occurring in latter compartments, suggesting that hydrogenotrophic methanogens were the primary methane generators in this bioreactor. However, aceticlastic methanogens could be involved in compartments related to the hydrolysis/acidogenesis stage.