Pan-Genomics of Lactobacillus plantarum Revealed Group-Specific Genomic Profiles without Habitat Association.

Lactobacillus plantarum is a lactic acid bacterium that promotes animal intestinal health as a probiotic and is found in a wide variety of habitats. Here, we investigated the genomic features of different clusters of L. plantarum strains via pan-genomic analysis. We compared the genomes of 108 L. plantarum strains that were available from the NCBI GenBank database. These genomes were 2.9-3.7 Mbp in size and 44-45% in G+C content. A total of 8,847 orthologs were collected, and 1,709 genes were identified to be shared as core genes by all the strains analyzed. On the basis of SNPs from the core genes, 108 strains were clustered into five major groups (G1-G5) that are different from previous reports and are not clearly associated with habitats. Analysis of group-specific enriched or depleted genes revealed that G1 and G2 were rich in genes for carbohydrate utilization (L-arabinose, L-rhamnose, and fructooligosaccharides) and that G3, G4, and G5 possessed more genes for the restriction-modification system and MazEF toxin-antitoxin. These results indicate that there are critical differences in gene content and survival strategies among genetically clustered L. plantarum strains, regardless of habitats.

Regulation of CD4+CD8-CD25+ and CD4+CD8+CD25+ T cells by gut microbiota in chicken.

The gut microbiota in chicken has long been studied, mostly from the perspective of growth performance. However, there are some immunological studies regarding gut homeostasis in chicken. Although CD4+CD25+ T cells are reported to act as regulatory T cells (Tregs) in chicken, there have been no studies showing the relationship between gut microbiota and Tregs. Therefore, we established a model for 'antibiotics (ABX)-treated chickens' through administration of an antibiotic cocktail consisting of ampicillin, gentamycin, neomycin, metronidazole, and vancomycin in water for 7 days. CD4+CD8-CD25+ and CD4+CD8+CD25+ T cells in cecal tonsils were significantly decreased in this model. Gram-positive bacteria, especially Clostridia, was responsible for the changes in CD4+CD8-CD25+ or CD4+CD8+CD25+ T cells in cecal tonsils. Feeding ABX-treated chickens with acetate recovered CD4+CD8-CD25+ and CD4+CD8+CD25+ T cells in cecal tonsils. GPR43, a receptor for acetate, was highly expressed in CD4+CD8-CD25+ T cells. In conclusion, our study demonstrated that the gut microbiota can regulate the population of CD4+CD8-CD25+ and CD4+CD8+CD25+ T cells, and that acetate is responsible for the induction of CD4+CD8-CD25+ T cells in cecal tonsils via GPR43.

Tracing of the fecal microbiota of commercial pigs at five growth stages from birth to shipment.

The intestinal microbiota affect various physiological traits of host animals such as brain development, obesity, age, and the immune system. In the swine industry, understanding the relationship between intestinal microbiota and growth stage is essential because growth stage is directly related to the feeding system of pigs, thus we studied the intestinal microbiota of 32 healthy pigs across five sows at 10, 21, 63, 93, and 147 d of ages. The intestinal microbiota were altered with growth of pigs and were separated into three distinct clusters. The relative abundance of several phyla and genera were significantly different between growth stages. We observed co-occurrence pattern of the intestinal microbiota at each growth stage. In addition, we predicted the functions of the intestinal microbiota and confirmed that several KEGG pathways were significantly different between growth stages. We also explored the relationship between the intestinal microbiota and innate factors such as the maternal effect and gender. When pigs were young, innate factors affected on construction of intestinal microbiota, however this tendency was disappeared with growth. Our findings broaden the understanding of microbial ecology, and the results will be used as a reference for investigating host-microbe interactions in the swine industry.

Prohibition of antibiotic growth promoters has affected the genomic profiles of Lactobacillus salivarius inhabiting the swine intestine.

After the introduction of a ban on the use of antibiotic growth promoters (AGPs) for livestock, the feeding environment, including the composition of animal intestinal microbiota, has changed rapidly. We hypothesized that the microbial genomes have also been affected by this legal prohibition, and investigated an important member of the swine gut microbiota, Lactobacillus salivarius, with a pan-genomic approach. Here, we isolated 21 L. salivarius strains composed of 6 strains isolated before the AGP prohibition (SBPs) and 15 strains isolated after the AGP prohibition (SAPs) at an interval of a decade, and the draft genomes were generated de novo. Several genomic differences between SBPs and SAPs were identified, although the number and function of antibiotic resistance genes were not different. SBPs showed larger genome size and a higher number of orthologs, as well as lower genetic diversity, than SAPs. SBPs had genes associated with the utilization of L-rhamnose and D-tagatose for energy production. Because these sugars are also used in exopolysaccharide (EPS) synthesis, we tried to identify differences in biofilm formation-associated genes. The genes for the production of EPSs and extracellular proteins were different in terms of amino acid sequences. Indeed, SAPs formed dense biofilm and survived better than SBPs in the swine intestinal environment. These results suggest that SAPs have evolved and adapted to protect themselves from new selection pressure of the swine intestinal microenvironment by forming dense biofilms, adopting a distinct antibiotic resistance strategy. This finding is particularly important to understand the evolutionary changes in host-microbe interaction and provide detailed insight for the development of effective probiotics for livestock.

Evaluating the association between body weight and the intestinal microbiota of weaned piglets via 16S rRNA sequencing.

Due to the ban on the use of antimicrobial growth promoters in livestock feeds, understanding the relationship between intestinal microbiota and the physiology of the host has become very important for improving livestock performance. In this study, we investigated the relationship between intestinal microbiota and body weights of weaned piglets. Lighter (n = 9) and heavier (n = 9) 9-week-old weaned piglets were selected from approximately one hundred individuals based on their body weights. Their fecal microbial communities were analyzed by sequencing the V4 region of the 16S rRNA gene. The microbial richness estimators of the heavier piglets were significantly higher than those of the lighter piglets. At the phylum level, the microbiota of the heavier group had significantly higher levels of Firmicutes and a higher Firmicutes-to-Bacteroidetes ratio than that of the lighter group. At the genus level, the levels of several genera, such as Anaerococcus and Lactococcus, were significantly different in the two groups. In particular, the lighter group had significantly higher levels of opportunistic pathogenic bacteria, such as Anaerotruncus and Bacteroides, compared with those of the heavier group. Moreover, the levels of bacteria expressing the components of several metabolic pathways were significantly different in the two groups. The microbiota of the heavier group had a significantly higher involvement in three KEGG pathways concerned with xenobiotic degradation than that of the lighter group. These results may provide insights into host-microbe interactions occurring in the piglet intestine and will be useful in establishing a strategy for improving growth performance in the swine industry.

Pan-Genomic Approaches in Lactobacillus reuteri as a Porcine Probiotic: Investigation of Host Adaptation and Antipathogenic Activity.

After the introduction of a ban on the use of antibiotic growth promoters (AGPs) for livestock, reuterin-producing Lactobacillus reuteri is getting attention as an alternative to AGPs. In this study, we investigated genetic features of L. reuteri associated with host specificity and antipathogenic effect. We isolated 104 L. reuteri strains from porcine feces, and 16 strains, composed of eight strains exhibiting the higher antipathogenic effect (group HS) and eight strains exhibiting the lower effect (group LS), were selected for genomic comparison. We generated draft genomes of the 16 isolates and investigated their pan-genome together with the 26 National Center for Biotechnology Information-registered genomes. L. reuteri genomes organized six clades with multi-locus sequence analysis, and the clade IV includes the 16 isolates. First, we identified six L. reuteri clade IV-specific genes including three hypothetical protein-coding genes. The three annotated genes encode transposases and cell surface proteins, indicating that these genes are the result of adaptation to the host gastrointestinal epithelia and that these host-specific traits were acquired by horizontal gene transfer. We also identified differences between groups HS and LS in the pdu-cbi-cob-hem gene cluster, which is essential for reuterin and cobalamin synthesis, and six genes specific to group HS are revealed. While the strains of group HS possessed all genes of this cluster, LS strains have lost many genes of the cluster. This study provides a deeper understanding of the relationship between probiotic properties and genomic features of L. reuteri.

Age-associated molecular changes are deleterious and may modulate life span through diet.

Transition through life span is accompanied by numerous molecular changes, such as dysregulated gene expression, altered metabolite levels, and accumulated molecular damage. These changes are thought to be causal factors in aging; however, because they are numerous and are also influenced by genotype, environment, and other factors in addition to age, it is difficult to characterize the cumulative effect of these molecular changes on longevity. We reasoned that age-associated changes, such as molecular damage and tissue composition, may influence life span when used in the diet of organisms that are closely related to those that serve as a dietary source. To test this possibility, we used species-specific culture media and diets that incorporated molecular extracts of young and old organisms and compared the influence of these diets on the life span of yeast, fruitflies, and mice. In each case, the "old" diet or medium shortened the life span for one or both sexes. These findings suggest that age-associated molecular changes, such as cumulative damage and altered dietary composition, are deleterious and causally linked with aging and may affect life span through diet.

Development of a Rapid Identification Method for the Differentiation of Enterococcus Species Using a Species-Specific Multiplex PCR Based on Comparative Genomics.

Enterococci are lactic acid bacteria that are commonly found in food and in animal gut. Since 16 S ribosomal RNA (rRNA) sequences, genetic markers for bacterial identification, are similar among several Enterococcus species, it is very difficult to determine the correct species based on only 16 S rRNA sequences. Therefore, we developed a rapid method for the identification of different Enterococcus species using comparative genomics. We compared 38 genomes of 13 Enterococcus species retrieved from the National Center of Biotechnology Information database and identified 25,623 orthologs. Among the orthologs, four genes were specific to four Enterococcus species (Enterococcus faecalis, Enterococcus faecium, Enterococcus hirae, and Enterococcus durans). We designed species-specific primer sets targeting the genes and developed a multiplex PCR using primer sets that could distinguish the four Enterococcus species among the nine strains of Enterococcus species that were available locally. The multiplex PCR method also distinguished the four species isolated from various environments, such as feces of chicken and cow, meat of chicken, cow, and pigs, and fermented soybeans (Cheonggukjang and Doenjang). These results indicated that our novel multiplex PCR using species-specific primers could identify the four Enterococcus species in a rapid and easy way. This method will be useful to distinguish Enterococcus species in food, feed, and clinical settings.

WITHDRAWN: Evaluating the association between body weight and the intestinal microbiota of weaned piglets via 16S rRNA sequencing.

This article has been removed: please see Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy). The publisher regrets that an error occurred due to a technical issue which led to the premature publication of this paper. This error bears no reflection on the article or its authors. The publisher apologizes to the authors and the readers for this unfortunate error.

Genomic Features and Niche-Adaptation of Enterococcus faecium Strains from Korean Soybean-Fermented Foods.

Certain strains of Enterococcus faecium contribute beneficially to human health and food fermentation. However, other E. faecium strains are opportunistic pathogens due to the acquisition of virulence factors and antibiotic resistance determinants. To characterize E. faecium from soybean fermentation, we sequenced the genomes of 10 E. faecium strains from Korean soybean-fermented foods and analyzed their genomes by comparing them with 51 clinical and 52 non-clinical strains of different origins. Hierarchical clustering based on 13,820 orthologous genes from all E. faecium genomes showed that the 10 strains are distinguished from most of the clinical strains. Like non-clinical strains, their genomes are significantly smaller than clinical strains due to fewer accessory genes associated with antibiotic resistance, virulence, and mobile genetic elements. Moreover, we identified niche-associated gene gain and loss from the soybean strains. Thus, we conclude that soybean E. faecium strains might have evolved to have distinctive genomic features that may contribute to its ability to thrive during soybean fermentation.

Complete genome sequence and SNPs of Raja pulchra (Rajiformes, Rajidae) mitochondria.

Mitochondrial genomes were sequenced from five Raja pulchra individuals, and single-nucleotide polymorphisms (SNPs) were identified by comparing previously announced sequences in this study. Total 117 SNPs were detected and they were present in 2 rRNA genes, 9 tRNA genes, 13 protein coding genes and non-coding region. One deleted polymorphic site, which was located in 16S rRNA gene, was observed in two individuals. Six polymorphic sites were non-synonymous SNPs, which were distributed in ND1, ND2, ATP6 and ND4 gene. Phylogenic analysis validated current taxa. The genome sequences of R. pulchra mitochondria could be comparable information for understanding species divergence and genomic variation among the populations.

Complete sequences of eastern water bat, Myotis petax (Chiroptera; Microchiroptera; Vespertilionidae) mitogenome.

Complete mitochondria genome sequences of myotis petax (Chiroptera; Microchiroptera; Vespertilionidae) were first identified in the present study. The sequences were obtained from the four individuals and composition of nucleotide AT and GC was about 64.58% and 35.42%, respectively. The lengths of mitogenomes were ranged from 17 296 to 17 299 bp. Total 51 variable sites were observed in the four mitogenomes and 38 sites were singleton polymorphic sites. Phylogenic study revealed that the species would have relatively closed evolutionary distance with m. macrodactylus rather than other species in the genus, myotis. Present study will provide important genomic materials supporting confirmation of taxon of species called bats, which is included in one of the largest orders among the mammals.

Influence of Flaxseed Oil on Fecal Microbiota, Egg Quality and Fatty Acid Composition of Egg Yolks in Laying Hens.

Although there have been many attempts to produce ω-3 fatty acid-rich eggs using alpha-linolenic acid (ALA) that is a popular fatty acid in the poultry feed industry, only limited knowledge about the effects of ALA-enriched diets on chicken fecal microbiota is currently available. Herein we examined the changes in the fecal microbiota composition, egg quality traits and fatty acid composition of the egg yolks of laying hens fed ALA-rich flaxseed oil for 8 weeks. The animals fed the experimental diets that contained 0 % (group C), 0.5 % (group T1), and 1.0 % (group T2) of flaxseed oil, respectively, and eggs and feces were obtained for the analyses. ω-3 fatty acids, including ALA, were increased in T1 and T2 compared with C. Furthermore, the freshness of eggs was improved with no side effects on the eggs. The diet also changed the fecal microbiota; Firmicutes was increased in T1 and T2 (48.6 to 83 and 79.6 %) and Bacteroidetes was decreased (40.2 to 8.8 and 4.2 %). Principal coordinate analysis revealed that Lactobacillus, among the 56 examined genera, was the most influenced bacterial group in terms of the fecal microbial community shifts. These results indicate that ALA-rich diets influenced both the egg and fecal microbiota in beneficial manners in laying hens although the association between the fatty acid composition of the egg yolk and the fecal microbiota was not clear. This study is a first step to understand the effect of flaxseed oil as well as intestinal microbiota of laying hens.

Draft Genome Sequence of Lactobacillus plantarum Strain SNU.Lp177 from Pig Feces in South Korea.

Herein we report a draft genome sequence for Lactobacillus plantarum SNU.Lp177, which was isolated from animal gut pig feces in South Korea. The draft genome of L. plantarum SNU.Lp177 contains 3,204,772 bp with a G+C content of 44.98% in 101 contigs (N50 = 116,595 bp).