Postbiotic-based recombinant receptor activator of NF-κB ligand enhanced oral vaccine efficiency in chicken.

Functional M cells are differentiated by receptor activator of NF-κB ligand (RANKL) and capture of luminal antigens to initiate immune responses. We aimed to use postbiotic-based recombinant chicken RANKL (cRANKL) to promote M cell differentiation and test the efficacy of oral vaccines. Chicks were divided into three groups that were administered phosphate-buffered saline (PBS), cell extracts of wild-type Lactococcus lactis subsp. lactis IL1403 (WT_CE), or cell extracts of recombinant L. lactis expressing cRANKL (cRANKL_CE). The expression of the M cell marker was measured, and the gut microbiome was profiled. The efficiency of the infectious bursal disease (IBD) vaccine was tested after 12 consecutive days of administering cRANKL_CE. The chickens that were administered cRANKL_CE (p = 0.038) had significantly higher Annexin A5 (ANXA5) mRNA expression levels than those in the PBS group (PBS vs. WT_CE, p = 0.657). In the gut microbiome analysis, no significant changes were observed. However, the relative abundance of Escherichia-Shigella was negatively correlated (r = - 0.43, p = 0.019) with ANXA5 mRNA expression in Peyer's patches. cRANKL_CE/IBD (p = 0.018) had significantly higher IBD-specific faecal IgA levels than PBS/IBD (PBS/IBD vs. WT_CE/IBD, p = 0.217). Postbiotic-based recombinant cRANKL effectively improved the expression of M cell markers and the efficiency of oral vaccines. No significant changes were observed in the gut microbiome after administration of postbiotic-based recombinant cRANKL. This strategy can be used for the development of feed additives and adjuvants. KEY POINTS: • Postbiotic-based recombinant cRANKL enhanced the expression of ANXA5 in chicken. • The relative abundance of Escherichia-Shigella was negatively correlated with ANXA5 expression. • Postbiotic-based recombinant cRANKL effectively improved the efficiency of oral vaccine.

Omega-3-Rich Fish-Oil-Influenced Mouse Gut Microbiome Shaped by Intermittent Consumption of Beef.

Beef consumption can provide various amino acids, lipids, vitamins, and minerals; however, excessive intake causes metabolic disorders and increases the probability of obesity, atherosclerosis, and colorectal cancer. The intake of omega-3 fatty acids can ameliorate metabolic disorders by lowering blood glucose and triglyceride levels. In the present study, we investigated the effect of omega-3-rich fish oil on body performance and the gut microbiome in a beef-rich diet. Four-week-old C57BL/6 mice were distributed into four groups (chow diet [Chow], chow with beef diet [Beef], chow with omega-3 diet [Cw3], and chow with beef and omega-3 diet [Bw3]). We observed that body weight was unaltered between groups, and serum triglyceride levels were reduced in the omega-3 supplemented groups. The beta diversity indices, unweighted UniFrac distance (P = 0.001), and Jaccard distance (P = 0.001) showed statistically significant differences, and the principal coordinates analysis plot showed a clear separation between groups. In addition, the taxonomic comparison revealed that beef consumption increased numerous potentially pathogenic bacteria, including Escherichia-Shigella, Mucispirillum, Helicobacter, and Desulfovibrio, which were decreased following omega-3 supplementation. Metabolic comparison based on 16S rRNA revealed that energy and glucose metabolism were higher in omega-3 supplemented groups. Our findings suggest that the omega-3 supplementation under intermittent beef consumption contributes to changes in the gut microbiome and microbial metabolic pathways.

Oral Immunization of Mice with Cell Extracts from Recombinant Lactococcus lactis Expressing SARS-CoV-2 Spike Protein.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread all over the world and became a pandemic that named coronavirus disease-2019 (COVID-19). At present, several intramuscular vaccines have been successfully developed and mass vaccination has progressed in many countries. The aim of the study is to develop and examine an oral vaccine against COVID-19 with recombinant Lactococcus lactis IL1403, a strain of lactic acid bacteria, expressing SARS-CoV-2 spike (S) protein receptor-binding domain (RBD) S1 subunit as an immunizing antigen. PBS or cell extracts from recombinant L. lactis were orally administered into mice (control VS treatment), and formation of antigen-specific antibodies and changes in the gut microbiome were analyzed. Intracellular antigen was detected, but its secretion was not successful. After immunization, antigen-specific serum IgG and fecal IgA levels were 1.5-fold (P = 0.002) and 1.4-fold (P = 0.016) higher in the immunized mice (treatment) than control, respectively. Gut microbiome profiles were clearly separated between the two groups when analyzed for beta diversity with overall similarity. At the genus level, while Coprococcus (P = 0.036) and unclassified genus of Ruminococcaceae (P = 0.037) in treatment were more abundant than control, rc4-4 (P = 0.013) and Stenotrophomonas (P = 0.021) were less abundant. Our results indicate that cell extract containing SARS-CoV-2 antigen can induce mice to produce antigen-specific antibodies without overall changes in the gut microbiome. This strategy may be useful for the development of other oral viral vaccines.

Insights into the Gut and Skin Microbiome of Freshwater Fish, Smelt (Hypomesus nipponensis).

Freshwater smelt (Hypomesus nipponensis) is a planktivorous fish found in the river of South Korea, Japan, China, and Russia. Because of its specific characteristics living in the cold temperature, this species is economically valuable in the various countries that held winter festival. The body size of the smelt is too small, so people consumed raw smelt during the winter festival sometimes. However, the microbial studies of smelt are nonexistent. Here, we characterized and compared the bacterial communities in the gut and skin of freshwater smelts. We amplified, sequenced, and analyzed the V4 regions of bacterial 16S rRNA genes from freshwater smelts. The microbial diversity in the skin (375 OTUs) was much greater than that in the gut (250 OTUs). At the phylum level, Proteobacteria (gut: 51.5%; skin: 52.9%), Firmicutes (gut: 30.6%; skin: 25.4%), Bacteroidetes (gut: 7.7%; skin: 14.7%), and Actinobacteria (gut: 5.2%; skin: 3.8%) were predominant in both organs. At the genus level, Sphingomonas (gut: 24.9%; skin: 4.4%, P < 0.01) was more abundant in the gut, whereas Acinetobacter (gut: 0.8%; skin: 11.8%, P = 0.02) and Pseudomonas (gut: 0.3%; skin: 2.1%, P = 0.01) were more abundant in the skin. Both beneficial (Lactobacillus) and harmful (Staphylococcus and Streptococcus) bacteria were detected in both organs, even under freshwater conditions. These results revealed that smelts have their own unique microbial communities in the gut and skin. Our findings broaden the understanding of planktivorous freshwater fish microbiomes and provide new insights into fish microbiomes for ensuring food safety.

Differences in microbiome and virome between cattle and horses in the same farm.

OBJECTIVE: The ecosystem of an animal farm is composed of various elements, such as animals, farmers, plants, feed, soil, and microorganisms. A domesticated animal's health is largely connected with the reservoir of bacteria and viruses in animal farms. Although a few studies have focused on exploring the gut microbiome of animals, communities of microbiota and viruses in feedlots have not been thoroughly investigated. METHODS: Here, we collected feces and dust samples (4 groups: cattle feces, C_F; horse feces, H_F; cattle dust, C_D; and horse dust, H_D) from cattle and horse farms sharing the same housing and investigated their microbiome/virome communities by Illumina sequencing. RESULTS: Dust groups (C_D and H_D) showed higher microbial diversity than feces groups (C_F and H_F) regardless of animal species. From the microbial community analysis, all the samples from the four groups have major phyla such as Proteobacteria (min 37.1% to max 42.8%), Firmicutes (19.1% to 24.9%), Bacteroidetes (10.6% to 22.1%), and Actinobacteria (6.1% to 20.5%). The abundance of Streptococcus, which commonly recognized as equine pathogens, was significantly higher in the horse group (H_D and H_F). Over 99% among the classified virome reads were classified as Caudovirales, a group of tailed bacteriophages, in all four groups. Foot-and-mouth disease virus and equine adenovirus, which cause deadly diseases in cattle and horse, respectively, were not detected. CONCLUSION: Our results will provide baseline information to understand different gut and environmental microbial ecology between two livestock species.

Tracing the spatio-temporal dynamics of endangered fin whales (Balaenoptera physalus) within baleen whale (Mysticeti) lineages: a mitogenomic perspective.

To explore the spatio-temporal dynamics of endangered fin whales (Balaenoptera physalus) within the baleen whale (Mysticeti) lineages, we analyzed 148 published mitochondrial genome sequences of baleen whales. We used a Bayesian coalescent approach as well as Bayesian inferences and maximum likelihood methods. The results showed that the fin whales had a single maternal origin, and that there is a significant correlation between geographic location and evolution of global fin whales. The most recent common female ancestor of this species lived approximately 9.88 million years ago (Mya). Here, North Pacific fin whales first appeared about 7.48 Mya, followed by a subsequent divergence in Southern Hemisphere approximately 6.63 Mya and North Atlantic about 4.42 Mya. Relatively recently, approximately 1.76 and 1.42 Mya, there were two additional occurrences of North Pacific populations; one originated from the Southern Hemisphere and the other from an uncertain location. The evolutionary rate of this species was 1.002 × 10-3 substitutions/site/My. Our Bayesian skyline plot illustrates that the fin whale population has the rapid expansion event since ~ 2.5 Mya, during the Quaternary glaciation stage. Additionally, this study indicates that the fin whale has a sister group relationship with humpback whale (Meganoptera novaeangliae) within the baleen whale lineages. Of the 16 genomic regions, NADH5 showed the most powerful signal for baleen whale phylogenetics. Interestingly, fin whales have 16 species-specific amino acid residues in eight mitochondrial genes: NADH2, COX2, COX3, ATPase6, ATPase8, NADH4, NADH5, and Cytb.

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.

Genome sequencing reveals insights into physiology and longevity of the naked mole rat.

The naked mole rat (Heterocephalus glaber) is a strictly subterranean, extraordinarily long-lived eusocial mammal. Although it is the size of a mouse, its maximum lifespan exceeds 30 years, making this animal the longest-living rodent. Naked mole rats show negligible senescence, no age-related increase in mortality, and high fecundity until death. In addition to delayed ageing, they are resistant to both spontaneous cancer and experimentally induced tumorigenesis. Naked mole rats pose a challenge to the theories that link ageing, cancer and redox homeostasis. Although characterized by significant oxidative stress, the naked mole rat proteome does not show age-related susceptibility to oxidative damage or increased ubiquitination. Naked mole rats naturally reside in large colonies with a single breeding female, the 'queen', who suppresses the sexual maturity of her subordinates. They also live in full darkness, at low oxygen and high carbon dioxide concentrations, and are unable to sustain thermogenesis nor feel certain types of pain. Here we report the sequencing and analysis of the naked mole rat genome, which reveals unique genome features and molecular adaptations consistent with cancer resistance, poikilothermy, hairlessness and insensitivity to low oxygen, and altered visual function, circadian rythms and taste sensing. This information provides insights into the naked mole rat's exceptional longevity and ability to live in hostile conditions, in the dark and at low oxygen. The extreme traits of the naked mole rat, together with the reported genome and transcriptome information, offer opportunities for understanding ageing and advancing other areas of biological and biomedical research.

Improved antimicrobial activity of Pediococcus acidilactici against Salmonella Gallinarum by UV mutagenesis and genome shuffling.

Pediococcus acidilactici is a widely used probiotic, and Salmonella enterica serovar Gallinarum (SG) is a significant pathogen in the poultry industry. In this study, we improved the antimicrobial activity of P. acidilactici against SG using UV mutation and genome shuffling (GS). To improve antimicrobial activity against SG, UV mutagenesis was performed against wild-type P. acidilactici (WT), and five mutants showed improved antimicrobial activity. To further improve antimicrobial activity, GS was performed on five UV mutants. Following GS, four mutants showed improved antimicrobial activity compared with the UV mutants and WT. The antimicrobial activity of GS1 was highest among the mutants; however, the activity was reduced when the culture supernatant was treated with proteinase K, suggesting that the improved antimicrobial activity is due to a proteinous substance such as bacteriocin. To validate the activity of GS1 in vivo, we designed multi-species probiotics and performed broiler feeding experiments. Groups consisted of no treatment (NC), avilamycin-treated (PC), probiotic group 1 containing WT (T1), and probiotic group 2 containing GS1 (T2). In broiler feeding experiments, coliform bacteria were significantly reduced in T2 compared with NC, PC, and T1. The cecal microbiota was modulated and pathogenic bacteria were reduced by GS1 oral administration. In this study, GS1 showed improved antimicrobial activity against SG in vitro and reduced pathogenic bacteria in a broiler feeding experiment. These results suggest that GS1 can serve as an efficient probiotic, as an alternative to antibiotics in the poultry industry.

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.

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.

Obese Mice Fed a Diet Supplemented with Enzyme-Treated Wheat Bran Display Marked Shifts in the Liver Metabolome Concurrent with Altered Gut Bacteria.

BACKGROUND: Enzyme-treated wheat bran (ETWB) contains a fermentable dietary fiber previously shown to decrease liver triglycerides (TGs) and modify the gut microbiome in mice. It is not clear which mechanisms explain how ETWB feeding affects hepatic metabolism, but factors (i.e., xenometabolites) associated with specific microbes may be involved. OBJECTIVE: The objective of this study was to characterize ETWB-driven shifts in the cecal microbiome and to identify correlates between microbial changes and diet-related differences in liver metabolism in diet-induced obese mice that typically display steatosis. METHODS: Five-week-old male C57BL/6J mice fed a 45%-lard-based fat diet supplemented with ETWB (20% wt:wt) or rapidly digestible starch (control) (n = 15/group) for 10 wk were characterized by using a multi-omics approach. Multivariate statistical analysis was used to identify variables that were strong discriminators between the ETWB and control groups. RESULTS: Body weight and liver TGs were decreased by ETWB feeding (by 10% and 25%, respectively; P < 0.001), and an index of liver reactive oxygen species was increased (by 29%; P < 0.01). The cecal microbiome showed an increase in Bacteroidetes (by 42%; P < 0.05) and a decrease in Firmicutes (by 16%; P < 0.05). Metabolites that were strong discriminators between the ETWB and control groups included decreased liver antioxidants (glutathione and α-tocopherol); decreased liver carbohydrate metabolites, including glucose; lower hepatic arachidonic acid; and increased liver and plasma ß-hydroxybutyrate. Liver transcriptomics revealed key metabolic pathways affected by ETWB, especially those related to lipid metabolism and some fed- or fasting-regulated genes. CONCLUSIONS: Together, these changes indicate that dietary fibers such as ETWB regulate hepatic metabolism concurrently with specific gut bacteria community shifts in C57BL/6J mice. It is proposed that these changes may elicit gut-derived signals that reach the liver via enterohepatic circulation, ultimately affecting host liver metabolism in a manner that mimics, in part, the fasting state.

Mice Fed a High-Fat Diet Supplemented with Resistant Starch Display Marked Shifts in the Liver Metabolome Concurrent with Altered Gut Bacteria.

BACKGROUND: High-amylose-maize resistant starch type 2 (HAMRS2) is a fermentable dietary fiber known to alter the gut milieu, including the gut microbiota, which may explain the reported effects of resistant starch to ameliorate obesity-associated metabolic dysfunction. OBJECTIVE: Our working hypothesis was that HAMRS2-induced microbiome changes alter gut-derived signals (i.e., xenometabolites) reaching the liver via the portal circulation, in turn altering liver metabolism by regulating gene expression and other pathways. METHODS: We used a multi-omics systems biology approach to characterize HAMRS2-driven shifts to the cecal microbiome, liver metabolome, and transcriptome, identifying correlates between microbial changes and liver metabolites under obesogenic conditions that, to our knowledge, have not previously been recognized. Five-week-old male C57BL/6J mice were fed an energy-dense 45% lard-based-fat diet for 10 wk supplemented with either 20% HAMRS2 by weight (n = 14) or rapidly digestible starch (control diet; n = 15). RESULTS: Despite no differences in food intake, body weight, glucose tolerance, fasting plasma insulin, or liver triglycerides, the HAMRS2 mice showed a 15-58% reduction in all measured liver amino acids, except for Gln, compared with control mice. These metabolites were equivalent in the plasma of HAMRS2 mice compared with controls, and transcripts encoding key amino acid transporters were not different in the small intestine or liver, suggesting that HAMRS2 effects were not simply due to lower hepatocyte exposure to systemic amino acids. Instead, alterations in gut microbial metabolism could have affected host nitrogen and amino acid homeostasis: HAMRS2 mice showed a 62% increase (P < 0.0001) in 48-h fecal output and a 41% increase (P < 0.0001) in fecal nitrogen compared with control mice. Beyond amino acid metabolism, liver transcriptomics revealed pathways related to lipid and xenobiotic metabolism; and pathways related to cell proliferation, differentiation, and growth were affected by HAMRS2 feeding. CONCLUSION: Together, these differences indicate that HAMRS2 dramatically alters hepatic metabolism and gene expression concurrent with shifts in specific gut bacteria in C57BL/6J mice.

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.

Overexpression of OCT4A ortholog elevates endogenous XIST in porcine parthenogenic blastocysts.

X-chromosome inactivation (XCI) is an epigenetic process that equalizes expression of X-borne genes between male and female eutherians. This process is observed in early eutherian embryo development in a species-specific manner. Until recently, various pluripotent factors have been suggested to regulate the process of XCI by repressing XIST expression, which is the master inducer for XCI. Recent insights into the process and its regulation have been restricted in mouse species despite the evolutionary diversity of the process and molecular mechanism among the species. OCT4A is one of the represented pluripotent factors, the gate-keeper for maintaining pluripotency, and an XIST repressor. Therefore, in here, we examined the relation between OCT4A and X-linked genes in porcine preimplantation embryos. Three X-linked genes, XIST, LOC102165544, and RLIM, were selected in present study because their orthologues have been known to regulate XCI in mice. Expression levels of OCT4A were positively correlated with XIST and LOC102165544 in female blastocysts. Furthermore, overexpression of exogenous human OCT4A in cleaved parthenotes generated blastocysts with increased XIST expression levels. However, increased XIST expression was not observed when exogenous OCT4A was obtained from early blastocysts. These results suggest the possibility that OCT4A would be directly or indirectly involved in XIST expression in earlier stage porcine embryos rather than blastocysts.

Nonclinical and clinical Enterococcus faecium strains, but not Enterococcus faecalis strains, have distinct structural and functional genomic features.

Certain strains of Enterococcus faecium and Enterococcus faecalis contribute beneficially to animal health and food production, while others are associated with nosocomial infections. To determine whether there are structural and functional genomic features that are distinct between nonclinical (NC) and clinical (CL) strains of those species, we analyzed the genomes of 31 E. faecium and 38 E. faecalis strains. Hierarchical clustering of 7,017 orthologs found in the E. faecium pangenome revealed that NC strains clustered into two clades and are distinct from CL strains. NC E. faecium genomes are significantly smaller than CL genomes, and this difference was partly explained by significantly fewer mobile genetic elements (ME), virulence factors (VF), and antibiotic resistance (AR) genes. E. faecium ortholog comparisons identified 68 and 153 genes that are enriched for NC and CL strains, respectively. Proximity analysis showed that CL-enriched loci, and not NC-enriched loci, are more frequently colocalized on the genome with ME. In CL genomes, AR genes are also colocalized with ME, and VF are more frequently associated with CL-enriched loci. Genes in 23 functional groups are also differentially enriched between NC and CL E. faecium genomes. In contrast, differences were not observed between NC and CL E. faecalis genomes despite their having larger genomes than E. faecium. Our findings show that unlike E. faecalis, NC and CL E. faecium strains are equipped with distinct structural and functional genomic features indicative of adaptation to different environments.

Safety of the surrogate microorganism Enterococcus faecium NRRL B-2354 for use in thermal process validation.

Enterococcus faecium NRRL B-2354 is a surrogate microorganism used in place of pathogens for validation of thermal processing technologies and systems. We evaluated the safety of strain NRRL B-2354 based on its genomic and functional characteristics. The genome of E. faecium NRRL B-2354 was sequenced and found to comprise a 2,635,572-bp chromosome and a 214,319-bp megaplasmid. A total of 2,639 coding sequences were identified, including 45 genes unique to this strain. Hierarchical clustering of the NRRL B-2354 genome with 126 other E. faecium genomes as well as pbp5 locus comparisons and multilocus sequence typing (MLST) showed that the genotype of this strain is most similar to commensal, or community-associated, strains of this species. E. faecium NRRL B-2354 lacks antibiotic resistance genes, and both NRRL B-2354 and its clonal relative ATCC 8459 are sensitive to clinically relevant antibiotics. This organism also lacks, or contains nonfunctional copies of, enterococcal virulence genes including acm, cyl, the ebp operon, esp, gelE, hyl, IS16, and associated phenotypes. It does contain scm, sagA, efaA, and pilA, although either these genes were not expressed or their roles in enterococcal virulence are not well understood. Compared with the clinical strains TX0082 and 1,231,502, E. faecium NRRL B-2354 was more resistant to acidic conditions (pH 2.4) and high temperatures (60°C) and was able to grow in 8% ethanol. These findings support the continued use of E. faecium NRRL B-2354 in thermal process validation of food products.

Short communication: effect of milk and milk containing Lactobacillus casei on the intestinal microbiota of mice.

BALB/c mice were fed milk or Lactobacillus casei BL23 in milk for 14d and fecal samples were collected at d 0, 4, and 7 as well as 1 and 8d after the last administration. According to high-throughput DNA sequencing of the 16S rRNA genes extracted from the fecal microbiota, the bacterial diversity in the fecal samples of all mice increased over time. After 14d of administration, the consumption of milk and milk containing L. casei BL23 resulted in distinct effects on the microbial composition in the intestine. Specifically, the proportions of bacteria in the Lactobacillaceae, Porphyromonadaceae, and Comamonadaceae were significantly higher in mice fed the L. casei BL23-milk culture compared with one or more of the other groups of mice. The relative amounts of Lachnospiraceae were higher and Streptococcaceae were lower in mice fed milk alone. The changes were not found at d 4 and 7 during milk and L. casei feeding and were no longer detected 8d after administration was stopped. This study shows that consumption of milk or probiotic L. casei-containing milk results in non-overlapping, taxa-specific effects on the bacteria in the distal murine intestine.

Effects of supplemental bacteriophage on the gut microbiota and nutrient digestibility of ileal-cannulated pigs.

This study measured the potential changes of the microbiota in the gastrointestinal tract and energy and nutrient digestibility by supplemental bacteriophages in pigs. Twelve castrated male pigs (initial mean body weight = 29.5 ± 2.3 kg) were surgically cannulated using T-cannula. The animals were housed individually in pens equipped with a feeder and a nipple waterer. The pigs were allotted to 1 of 3 experimental diets in a quadruplicated 3 × 2 Latin square design with 3 experimental diets, 2 periods, and 12 pigs resulting in 8 replicates per diet. The 3 diets were a control mainly based on corn and soybean meal with no antibiotics or bacteriophages, a diet containing 0.1% antibiotics, and a diet containing 0.2% bacteriophages. On day 5 of the experimental period, feces were collected and on days 6 and 7, ileal digesta were collected. Genomic DNA for bacteria were extracted from the ileal digesta and feces and the V4 region of the 16S rRNA gene was amplified. The ileal and fecal digestibility of energy, dry matter, organic matter, crude protein, and fiber was unaffected by dietary antibiotics or bacteriophages. At the phylum level, the supplemental antibiotic or bacteriophage tended to result in a higher proportion of Firmicutes (p = 0.059) and a lower proportion of Bacteroidetes (p = 0.099) in the ileal digesta samples compared with the control group with no difference between the antibiotic and bacteriophage groups. At the genus level, the supplemental antibiotic or bacteriophage tended to result in a higher proportion of Lactobacillus (p = 0.062) and a lower proportion of Bacteroides (p = 0.074) and Streptococcus (p = 0.088) in the ileal digesta compared with the control group with no difference between the antibiotic and bacteriophage groups. In the feces, supplemental antibiotics or bacteriophages reduced the proportion of Bifidobacterium compared with the control group (p = 0.029) with no difference between the antibiotic and bacteriophage groups. Overall, supplemental antibiotics and bacteriophages showed positive effect on the microbiota of in the ileal digesta without largely affecting energy or nutrient digestibility, with no differences between the antibiotic and bacteriophage groups in growing pigs.

A comprehensive longitudinal study of gut microbiota dynamic changes in laying hens at four growth stages prior to egg production.

OBJECTIVE: The poultry industry is a primary source of animal protein worldwide. The gut microbiota of poultry birds, such as chickens and ducks, is critical in maintaining their health, growth, and productivity. This study aimed to identify longitudinal changes in the gut microbiota of laying hens from birth to the pre-laying stage. METHODS: From a total of 80 Hy-Line Brown laying hens, birds were selected based on weight at equal intervals to collect feces (n = 20 per growth) and ileal contents (n = 10 per growth) for each growth stage (days 10, 21, 58, and 101). The V4 regions of the 16S rRNA gene were amplified after extracting DNA from feces and ileal contents. Amplicon sequencing was performed using Illumina, followed by analysis. RESULTS: Microbial diversity increased with growth stages, regardless of sampling sites. Microbial community analysis indicated that Firmicutes, Proteobacteria, and Bacteroidetes were the dominant phyla in the feces and ileal. The abundance of Lactobacillus was highest on day 10, and that of Escherichia-shigella was higher on day 21 than those at the other stages at the genus level (for the feces and ileal contents; p<0.05). Furthermore, Turicibacter was the most abundant genus after changing feed (for the feces and ileal contents; p<0.05). The fecal Ruminococcus torques and ileal Lysinibacillus were negatively correlated with the body weights of chickens (p<0.05). CONCLUSION: The gut microbiota of laying hens changes during the four growth stages, and interactions between microbiota and feed may be present. Our findings provide valuable data for understanding the gut microbiota of laying hens at various growth stages and future applied studies.