The diversity of the equine fecal bacterial community was evaluated using pyrosequencing of 16S rRNA gene amplicons. Fecal samples were obtained from horses fed cool-season grass hay. Fecal bacteria were characterized by amplifying the V4 region of bacterial 16S rRNA gene. Of 5898 mean unique sequences, a mean of 1510 operational taxonomic units were identified in the four fecal samples. Equine fecal bacterial richness was higher than that reported in humans, but lower than that reported in either cattle feces or soil. Bacterial classified sequences were assigned to 16 phyla, of which 10 were present in all samples. The largest number of reads belonged to Firmicutes (43.7% of total bacterial sequences), Verrucomicrobia (4.1%), Proteobacteria (3.8%), and Bacteroidetes (3.7%). The less abundant Actinobacteria, Cyanobacteria, and TM7 phyla presented here have not been previously described in the gut contents or feces of horses. Unclassified sequences represented 38.1% of total bacterial sequences; therefore, the equine fecal microbiome diversity is likely greater than that described. This is the first study to characterize the fecal bacterial community in horses by the use of 16S rRNA gene amplicon pyrosequencing, expanding our knowledge of the fecal microbiota of forage-fed horses.

Aims:  To investigate the changes in bacterial diversity on fresh spinach phyllosphere associated with storage at refrigeration temperatures. Methods and Results:  Community structure and population dynamics of spinach phylloepiphytic bacteria associated with packaging and refrigeration of ready‐to‐eat fresh produce were evaluated using pyrosequencing of 16S rRNA gene amplicons. A diverse community responsive to storage at refrigerated temperatures was detected belonging to over 1000 operational taxonomic units, including many diverse members not previously described on the phyllosphere. Of the approx. 8800 unique sequences examined from fresh spinach leaves, 75% were from previously undescribed taxa. The classified sequences from the fresh spinach phyllosphere were assigned to 11 different phyla with the largest number of reads belonging to Proteobacteria and Firmicutes. Packaging and storage of spinach under refrigerated conditions decreased the richness, diversity and evenness of the bacterial community. Refrigeration at 4 and 10°C and storage resulted in a decrease in number of taxa represented from 11 phyla in fresh spinach to only 5 phyla after 1 day of storage. Sequences belonging to γ‐Proteobacteria, particularly Pseudomonas spp. and members of the Enterobacteriaceae, were the most numerous after 15 days of storage at both temperatures. Growth inhibition of the genera Escherichia was achieved at 4°C but not at 10°C storage, thus highlighting the importance of temperature in fresh packaged spinach. Conclusions:  The application of pyrosequencing to describe composition and diversity of the phyllosphere on spinach leaves provided a broader outlook of the bacterial composition of this community complementing other phyllosphere studies that have used culture‐ and nonculture‐dependent approaches. Significance and Impact of the Study:  Pyrosequencing allowed a broader description of the bacterial composition and diversity of the spinach leaf surface than previously obtained using culture‐based detection and will be a powerful tool to help ensure the future safety and quality of packaged spinach.

The microbial composition of ancient permafrost sediments from the Kolyma lowland of Northeast Eurasia was examined through culture and culture-independent approaches. These sediments have been continuously frozen for 5,000 to 2-3 million years. A total of 265 Bacteria 16S rRNA gene sequences were amplified from the permafrost total-community genomic DNA and screened by amplified ribosomal 16S rRNA restriction analysis. Members of three major lineages were found: gamma-Proteobacteria (mostly Xanthomonadaceae), Actinobacteria, and Firmicutes. We also determined partial 16S rRNA gene sequences of 49 isolates from a collection of 462 aerobes isolated from these sediments. The bacteria included Actinomycetales (Arthrobacter and Microbacteriaceae); followed by the Firmicutes (Exiguobacterium and Planomicrobium); the Bacteroidetes (Flavobacterium); the gamma-Proteobacteria (Psychrobacter); and the alpha-Proteobacteria (Sphingomonas). Both culture and culture-independent approaches showed the presence of high and low G+C Gram-positive bacteria and gamma-Proteobacteria. Some of the 16S rRNA gene sequences of environmental clones matched those of Arthrobacter isolates. Two-thirds of the isolates grew at -2.5 degrees C, indicating that they are psychroactive, and all are closely related to phylogenetic groups with strains from other cold environments, mostly commonly from Antarctica. The culturable and non-culturable microorganisms found in the terrestrial permafrost provide a prototype for possible life on the cryogenic planets of the Solar System.

Psychrobacter arcticus strain 273-4, which grows at temperatures as low as ؊10°C, is the first cold-adapted bacterium from a terrestrial environment whose genome was sequenced. Analysis of the 2.65-Mb genome suggested that some of the strategies employed by P. arcticus 273-4 for survival under cold and stress conditions are changes in membrane composition, synthesis of cold shock proteins, and the use of acetate as an energy source. Comparative genome analysis indicated that in a significant portion of the P. arcticus proteome there is reduced use of the acidic amino acids and proline and arginine, which is consistent with increased protein flexibility at low temperatures. Differential amino acid usage occurred in all gene categories, but it was more common in gene categories essential for cell growth and reproduction, suggesting that P. arcticus evolved to grow at low temperatures. Amino acid adaptations and the gene content likely evolved in response to the long-term freezing temperatures (؊10°C to ؊12°C) of the Kolyma (Siberia) permafrost soil from which this strain was isolated. Intracellular water likely does not freeze at these in situ temperatures, which allows P. arcticus to live at subzero temperatures.

Three Gram-negative, non-motile, non-pigmented, oxidase-positive coccobacilli capable of growth at temperatures from −10 to 30 °C and salinities of 0 to 1.7 M NaCl were isolated from Siberian permafrost and characterized. Both 16S rRNA and gyrB gene sequencing studies placed the isolates in the Gammaproteobacteria within the genus Psychrobacter. However, with higher bootstrap values and reproducible tree topologies, gyrB represented a more reliable phylogenetic marker for the taxonomy of Psychrobacter species. DNA–DNA hybridization data supported gyrB tree topologies and established two relatedness groups within the three isolates; neither of these groups was related at the species level to any previously described Psychrobacter species. The two groups of isolates could be differentiated phenotypically from 13 previously described Psychrobacter species using API strips. These results support the existence of two novel species of Psychrobacter, for which we propose the names Psychrobacter cryohalolentis sp. nov. (type strain K5T=DSM 17306T=VKM B-2378T) and Psychrobacter arcticus sp. nov. (type strain 273-4T=DSM 17307T=VKM B-2377T).

Past studies of cold-acclimated bacteria have focused primarily on organisms not capable of sub-zero growth. Siberian permafrost isolates Exiguobacterium sp. 255-15 and Psychrobacter sp. 273-4, which grow at subzero temperatures, were used to study cold-acclimated physiology. Changes in membrane composition and exopolysaccharides were defined as a function of growth at 24, 4 and -2.5 degrees C in the presence and absence of 5% NaCl. As expected, there was a decrease in fatty acid saturation and chain length at the colder temperatures and a further decrease in the degree of saturation at higher osmolarity. A shift in carbon source utilization and antibiotic resistance occurred at 4 versus 24 degrees C growth, perhaps due to changes in the membrane transport. Some carbon substrates were used uniquely at 4 degrees C and, in general, increased antibiotic sensitivity was observed at 4 degrees C. All the permafrost strains tested were resistant to long-term freezing (1 year) and were not particularly unique in their UVC tolerance. Most of the tested isolates had moderate ice nucleation activity, and particularly interesting was the fact that the Gram-positive Exiguobacterium showed some soluble ice nucleation activity. In general the features measured suggest that the Siberian organisms have adapted to the conditions of long-term freezing at least for the temperatures of the Kolyma region which are -10 to -12 degrees C where intracellular water is likely not frozen.

Walnuts are rich in omega-3 fatty acids, phytochemicals and antioxidants making them unique compared to other foods. Consuming walnuts has been associated with health benefits including a reduced risk of heart disease and cancer. Dysbiosis of the gut microbiome has been linked to several chronic diseases. One potential mechanism by which walnuts may exert their health benefit is through modifying the gut microbiome. This study identified the changes in the gut microbial communities that occur following the inclusion of walnuts in the diet. Male Fischer 344 rats (n=20) were randomly assigned to one of two diets for as long as 10 weeks: (1) walnut (W), and (2) replacement (R) in which the fat, fiber, and protein in walnuts were matched with corn oil, protein casein, and a cellulose fiber source. Intestinal samples were collected from the descending colon, the DNA isolated, and the V3-V4 hypervariable region of 16S rRNA gene deep sequenced on an Illumina MiSeq for characterization of the gut microbiota. Body weight and food intake did not differ significantly between the two diet groups. The diet groups had distinct microbial communities with animals consuming walnuts displaying significantly greater species diversity. Walnuts increased the abundance of Firmicutes and reduced the abundance of Bacteriodetes. Walnuts enriched the microbiota for probiotic-type bacteria including Lactobacillus, Ruminococcaceae, and Roseburia while significantly reducing Bacteroides and Anaerotruncus. The class Alphaproteobacteria was also reduced. Walnut consumption altered the gut microbial community suggesting a new mechanism by which walnuts may confer their beneficial health effects.

Anaerobically fermented yeast products are a rich source of nutritional metabolites, mannanoligosaccharides, and β-glucans that may optimize gut health and immunity, which can translate into better growth performance and a reduced risk of foodborne pathogens. The objective of this study was to quantify the effects of Saccharomyces cerevisiae fermentation product (Diamond V Original XPC) inclusion in nursery diets on pig performance and gastrointestinal microbial ecology before, during, and after an oral challenge with Salmonella. Pigs (n = 40) were weaned at 21 d of age, blocked by BW, and assigned in a 2 × 2 factorial arrangement consisting of diet (control or 0.2% XPC) and inoculation (sterile broth or Salmonella). Pigs were fed a 3-phase nursery diet (0 to 7 d, 7 to 21 d, and 21 to 35 d) with ad libitum access to water and feed. On d 14, pigs were orally inoculated with 10(9) cfu of Salmonella enterica serovar Typhimurium DT104 or sterile broth. During d 17 to 20, all pigs were treated with a 5 mg/kg of BW intramuscular injection of ceftiofur-HCl. Growth performance and alterations in the gastrointestinal microbial ecology were measured during preinoculation (PRE; 0 to 14 d), sick (SCK; 14 to 21 d), and postinoculation (POST; 21 to 35 d). Body weight and ADG were measured weekly. Rectal temperature (RT) was measured weekly during PRE and POST, and every 12 h during SCK. Diet had no effect on BW, ADG, or RT during any period (P = 0.12 to 0.95). Inclusion of XPC tended (P < 0.10) to increase Salmonella shedding in feces during SCK. Consumption of XPC altered the composition of the gastrointestinal microbial community, resulting in increased (P < 0.05) populations of Bacteroidetes and Lactobacillus after Salmonella infection. Pigs inoculated with Salmonella had decreased ADG and BW, and increased RT during SCK (P < 0.001). Furthermore, fecal Salmonella cfu (log(10)) was modestly correlated (P = 0.002) with BW (r = -0.22), ADFI (r = -0.27), ADG (r = -0.36), G:F (r = -0.18), and RT (r = 0.52) during SCK. After antibiotic administration, all Salmonella-infected pigs stopped shedding. During POST, an interaction between diet and inoculation (P = 0.009) on ADG indicated that pigs infected with Salmonella grew better when eating XPC than the control diet. The addition of XPC to the diets of weanling pigs resulted in greater compensatory BW gains after infection with Salmonella than in pigs fed conventional nursery diets. This increase in BW gain is likely associated with an increase in beneficial bacteria within the gastrointestinal tract.