Characterizing the gut phageome and phage-borne antimicrobial resistance genes in pigs.

BACKGROUND: Mammalian intestine harbors a mass of phages that play important roles in maintaining gut microbial ecosystem and host health. Pig has become a common model for biomedical research and provides a large amount of meat for human consumption. However, the knowledge of gut phages in pigs is still limited. RESULTS: Here, we investigated the gut phageome in 112 pigs from seven pig breeds using PhaBOX strategy based on the metagenomic data. A total of 174,897 non-redundant gut phage genomes were assembled from 112 metagenomes. A total of 33,487 gut phage genomes were classified and these phages mainly belonged to phage families such as Ackermannviridae, Straboviridae, Peduoviridae, Zierdtviridae, Drexlerviridae, and Herelleviridae. The gut phages in seven pig breeds exhibited distinct communities and the gut phage communities changed with the age of pig. These gut phages were predicted to infect a broad range of 212 genera of prokaryotes, such as Candidatus Hamiltonella, Mycoplasma, Colwellia, and Lactobacillus. The data indicated that broad KEGG and CAZy functions were also enriched in gut phages of pigs. The gut phages also carried the antimicrobial resistance genes (ARGs) and the most abundant antimicrobial resistance genotype was diaminopyrimidine resistance. CONCLUSIONS: Our research delineates a landscape for gut phages in seven pig breeds and reveals that gut phages serve as a key reservoir of ARGs in pigs. Video Abstract.

Characterizing core microbiota and regulatory functions of the pig gut microbiome.

Domestic pigs (Sus scrofa) are the leading terrestrial animals used for meat production. The gut microbiota significantly affect host nutrition, metabolism, and immunity. Hence, characterization of the gut microbial structure and function will improve our understanding of gut microbial resources and the mechanisms underlying host-microbe interactions. Here, we investigated the gut microbiomes of seven pig breeds using metagenomics and 16S rRNA gene amplicon sequencing. We established an expanded gut microbial reference catalog comprising 17 020 160 genes and identified 4910 metagenome-assembled genomes. We also analyzed the gut resistome to provide an overview of the profiles of the antimicrobial resistance genes in pigs. By analyzing the relative abundances of microbes, we identified three core-predominant gut microbes (Phascolarctobacterium succinatutens, Prevotella copri, and Oscillibacter valericigenes) in pigs used in this study. Oral administration of the three core-predominant gut microbes significantly increased the organ indexes (including the heart, spleen, and thymus), but decreased the gastrointestinal lengths in germ-free mice. The three core microbes significantly enhanced intestinal epithelial barrier function and altered the intestinal mucosal morphology, as was evident from the increase in crypt depths in the duodenum and ileum. Furthermore, the three core microbes significantly affected several metabolic pathways (such as "steroid hormone biosynthesis," "primary bile acid biosynthesis," "phenylalanine, tyrosine and tryptophan biosynthesis," and "phenylalanine metabolism") in germ-free mice. These findings provide a panoramic view of the pig gut microbiome and insights into the functional contributions of the core-predominant gut microbes to the host.

Ghrelin in Focus: Dissecting Its Critical Roles in Gastrointestinal Pathologies and Therapies.

This review elucidates the critical role of ghrelin, a peptide hormone mainly synthesized in the stomach in various gastrointestinal (GI) diseases. Ghrelin participates in diverse biological functions ranging from appetite regulation to impacting autophagy and apoptosis. In sepsis, it reduces intestinal barrier damage by inhibiting inflammatory responses, enhancing GI blood flow, and modulating cellular processes like autophagy and apoptosis. Notably, in inflammatory bowel disease (IBD), serum ghrelin levels serve as markers for distinguishing between active and remission phases, underscoring its potential in IBD treatment. In gastric cancer, ghrelin acts as an early risk marker, and due to its significant role in increasing the proliferation and migration of gastric cancer cells, the ghrelin-GHS-R axis is poised to become a target for gastric cancer treatment. The role of ghrelin in colorectal cancer (CRC) remains controversial; however, ghrelin analogs have demonstrated substantial benefits in treating cachexia associated with CRC, highlighting the therapeutic potential of ghrelin. Nonetheless, the complex interplay between ghrelin's protective and potential tumorigenic effects necessitates a cautious approach to its therapeutic application. In post-GI surgery scenarios, ghrelin and its analogs could be instrumental in enhancing recovery and reducing complications. This article accentuates ghrelin's multifunctionality, shedding light on its influence on disease mechanisms, including inflammatory responses and cancer progression, and examines its therapeutic potential in GI surgeries and disorders, advocating for continued research in this evolving field.

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A growing body of evidence has linked the gut microbiota to liver metabolism. The manipulation of intestinal microflora has been considered as a promising avenue to promote liver health. However, the effects of Lactobacillus gasseri LA39, a potential probiotic, on liver metabolism remain unclear. Accumulating studies have investigated the proteomic profile for mining the host biological events affected by microbes, and used the germ-free (GF) mouse model to evaluate host-microbe interaction. Here, we explored the effects of L. gasseri LA39 gavage on the protein expression profiles of the liver of GF mice. Our results showed that a total of 128 proteins were upregulated, whereas a total of 123 proteins were downregulated by treatment with L. gasseri LA39. Further bioinformatics analyses suggested that the primary bile acid (BA) biosynthesis pathway in the liver was activated by L. gasseri LA39. Three differentially expressed proteins (cytochrome P450 family 27 subfamily A member 1 (CYP27A1), cytochrome P450 family 7 subfamily B member 1 (CYP7B1), and cytochrome P450 family 8 subfamily B member 1 (CYP8B1)) involved in the primary BA biosynthesis pathway were further validated by western blot assay. In addition, targeted metabolomic analyses demonstrated that serum and fecal ß|-muricholic acid (a primary BA), dehydrolithocholic acid (a secondary BA), and glycolithocholic acid-3-sulfate (a secondary BA) were significantly increased by L. gasseri LA39. Thus, our data revealed that L. gasseri LA39 activates the hepatic primary BA biosynthesis and promotes the intestinal secondary BA biotransformation. Based on these findings, we suggest that L. gasseri LA39 confers an important function in the gut‒liver axis through regulating BA metabolism.

Gut microbiota-derived 3-phenylpropionic acid promotes intestinal epithelial barrier function via AhR signaling.

BACKGROUND: The intestinal epithelial barrier confers protection against the intestinal invasion by pathogens and exposure to food antigens and toxins. Growing studies have linked the gut microbiota to the intestinal epithelial barrier function. The mining of the gut microbes that facilitate the function of intestinal epithelial barrier is urgently needed. RESULTS: Here, we studied a landscape of the gut microbiome of seven pig breeds using metagenomics and 16S rDNA gene amplicon sequencing. The results indicated an obvious difference in the gut microbiome between Congjiang miniature (CM) pigs (a native Chinese breed) and commercial Duroc × [Landrace × Yorkshire] (DLY) pigs. CM finishing pigs had stronger intestinal epithelial barrier function than the DLY finishing pigs. Fecal microbiota transplantation from CM and DLY finishing pigs to germ-free (GF) mice transferred the intestinal epithelial barrier characteristics. By comparing the gut microbiome of the recipient GF mice, we identified and validated Bacteroides fragilis as a microbial species that contributes to the intestinal epithelial barrier. B. fragilis-derived 3-phenylpropionic acid metabolite had an important function on the enhancement of intestinal epithelial barrier. Furthermore, 3-phenylpropionic acid facilitated the intestinal epithelial barrier by activating aryl hydrocarbon receptor (AhR) signaling. CONCLUSIONS: These findings suggest that manipulation of B. fragilis and 3-phenylpropionic acid is a promising strategy for improving intestinal epithelial barrier. Video Abstract.

Core-predominant gut fungus Kazachstania slooffiae promotes intestinal epithelial glycolysis via lysine desuccinylation in pigs.

BACKGROUND: Gut fungi are increasingly recognized as important contributors to host physiology, although most studies have focused on gut bacteria. Post-translational modifications (PTMs) of proteins play vital roles in cell metabolism. However, the contribution of gut fungi to host protein PTMs remains unclear. Mining gut fungi that mediate host protein PTMs and dissecting their mechanism are urgently needed. RESULTS: We studied the gut fungal communities of 56 weaned piglets and 56 finishing pigs from seven pig breeds using internal transcribed spacer (ITS) gene amplicon sequencing and metagenomics. The results showed that Kazachstania slooffiae was the most abundant gut fungal species in the seven breeds of weaned piglets. K. slooffiae decreased intestinal epithelial lysine succinylation levels, and these proteins were especially enriched in the glycolysis pathway. We demonstrated that K. slooffiae promoted intestinal epithelial glycolysis by decreasing lysine succinylation by activating sirtuin 5 (SIRT5). Furthermore, K. slooffiae-derived 5'-methylthioadenosine metabolite promoted the SIRT5 activity. CONCLUSIONS: These findings provide a landscape of gut fungal communities of pigs and suggest that K. slooffiae plays a crucial role in intestinal glycolysis metabolism through lysine desuccinylation. Our data also suggest a potential protective strategy for pigs with an insufficient intestinal energy supply. Video Abstract.

Ghrelin/GHSR Axis Induced M2 Macrophage and Alleviated Intestinal Barrier Dysfunction in a Sepsis Rat Model by Inactivating E2F1/NF-κB Signaling.

Sepsis is an inflammatory reaction disorder state that is induced by infection. The activation and regulation of the immune system play an essential role in the development of sepsis. Our previous studies have shown that ghrelin ameliorates intestinal dysfunction in sepsis. Very little is known about the mechanism of ghrelin and its receptor (GHSR) on the intestinal barrier and the immune function of macrophage regulation. Our research is to investigate the regulatory effect and molecular mechanism of the ghrelin/GHSR axis on intestinal dysfunction and macrophage polarization in septic rats. A rat model of sepsis was established by cecal ligation and puncture (CLP) operation. Then, the sepsis rats were treated with a ghrelin receptor agonist (TZP-101) or ghrelin inhibitor (obestatin). The results suggested that TZP-101 further enhanced ghrelin and GHSR expressions in the colon and spleen of septic rats and obestatin showed the opposite results. Ghrelin/GHSR axis ameliorated colonic structural destruction and intestinal epithelial tight junction injury in septic rats. In addition, the ghrelin/GHSR axis promoted M2-type polarization of macrophages, which was characterized by the decreases of IL-1ß, IL-6, and TNF-α, as well as the increase of IL-10. Mechanistically, the ghrelin/GHSR axis promoted E2F2 expression and suppressed the activation of the NF-κB signaling pathway in septic rats. Collectively, targeting ghrelin/GHSR during sepsis may represent a novel therapeutic approach for the treatment of intestinal barrier injury.

Lactobacillus frumenti improves antioxidant capacity via nitric oxide synthase 1 in intestinal epithelial cells.

Oxidative damages have adverse effects on mammals. Growing studies have focused on exploring new antioxidants. Here, we report that Lactobacillus frumenti increases the total antioxidation capacity activities and decreases the total reactive oxygen species levels in porcine intestinal epithelial cells. Comparative proteomics revealed that expressions of peroxiredoxin 2, isocitrate dehydrogenase 1, NAD(P)H dehydrogenase quinone 1, antioxidant protein 1, and metallothionein-2A, which are associated with antioxidant defense system, were significantly increased with L. frumenti treatment. In germ-free mice, L. frumenti treatment also remarkably improves the intestinal antioxidant capacity. We further illustrated that nitric oxide production-mediated by nitric oxide synthase 1 activation is essential for L. frumenti-induced improvements in intestinal epithelial antioxidant capacity and barrier function. This study suggested that L. frumenti may be a potential probiotic used to prevent oxidative stress-induced aging and diseases in mammals.-Nie, Y., Hu, J., Hou, Q., Zheng, W., Zhang, X., Yang, T., Ma, L., Yan, X. Lactobacillus frumenti improves antioxidant capacity via nitric oxide synthase 1 in intestinal epithelial cells.

Clinical efficacy of high-flow nasal humidified oxygen therapy in patients with hypoxemia.

To evaluate the effectiveness of high-flow nasal humidified oxygen (HFNHO) therapy in patients with mild hypoxemia after extubation. This study included 316 patients with mild hypoxemia after extubation from May 2016 to May 2018 from two intensive care units in China. Compare the effects of the Venturi Mask and High-Flow Nasal Humidified Oxygen (HFNHO) therapy on Heart Rate (HR), Respiratory Rate (RR), Oxygen Saturation (SpO2), Oxygen Partial Pressure (PO2), Partial Pressure Of Carbon Dioxide (PCO2), Oxygenation Index (PO2/FiO2) after extubation, the use of noninvasive mechanical ventilation and tracheal intubation after treatment failure were observed and recorded. Patients have both lower HR and RR than those who received mask treatment (75.4±18.5 vs. 83.0±20.4, p = 0.0004; 18±6.5 vs. 23.6±10.3, p<0.001, respectively). There was significant difference between those who had HFNHO and mask administration's SpO2 and PO2 (94.1±6.4 vs. 87.5±1.5, p<0.001; 88.16±2.9 vs. 77.3±2.3, p<0.001, respectively). For the HFNHO group, patients had lower PCO2 with the mask group. (41.3±0.99 vs 42.2±1.2, p<0.001). On the other hand, the levels of PO2/FiO2 was significantly higher in the HFNHO Group, (181.0±8.3 vs. 157.2±4.9, p<0.05). We concluded HFNHO therapy could significantly relieve the symptoms of dyspnea, improve oxygenation, reduce the use of noninvasive mechanical ventilation and reduce the rate of secondary tracheal intubation in patients with mild hypoxemia after extubation.

Alkannin attenuates lipopolysaccharide-induced lung injury in mice via Rho/ROCK/NF-κB pathway.

We investigated the effects and associated mechanism of alkannin (AL) on lipopolysaccharide (LPS)-induced acute lung injury in a mouse model. Pretreatment with AL in vivo significantly reduced the lung wet/dry weight ratio and inhibited lung myeloperoxidase activity and malondialdehyde content, while increasing superoxide dismutase activity. Hematoxylin and eosin staining demonstrated that AL attenuated lung histopathological changes. In addition, AL-inhibited overproduction of proinflammatory cytokines in bronchoalveolar lavage fluid and lung tissues in LPS-injured mice and LPS-exposed A549 cells. Further analysis showed that AL-inhibited induction of the Rho/ROCK/NF-κB pathway via LPS-induced inflammation in mice and A549 cells. Fasudil, a selective ROCK inhibitor, showed similar effects. Overall, the findings indicate that AL suppresses the expression of messenger RNAs and proteins associated with Rho/ROCK/NF-κB signaling to effectively ameliorate lung injury.

A Microbiota-Derived Bacteriocin Targets the Host to Confer Diarrhea Resistance in Early-Weaned Piglets.

Alternatives to antibiotics for preventing diarrhea in early-weaned farm animals are sorely needed. CM piglets (a native Chinese breed) are more resistant to early-weaning stress-induced diarrhea than the commercial crossbred LY piglets. Transferring fecal microbiota, but not saline, from healthy CM into LY piglets by oral administration prior to early weaning conferred diarrhea resistance. By comparing the relative abundance of intestinal microbiota in saline and microbiota transferred LY piglets, we identified and validated Lactobacillus gasseri LA39 and Lactobacillus frumenti as two bacterial species that mediate diarrhea resistance. Diarrhea resistance depended on the bacterial secretory circular peptide gassericin A, a bacteriocin. The binding of gassericin A to Keratin 19 (KRT19) on the plasma membrane of intestinal epithelial cells was essential for enhancement of fluid absorption and decreased secretion. These findings suggest the use of L. gasseri LA39 and L. frumenti as antibiotic alternatives for preventing diarrhea in mammals.

Standardized Preparation for Fecal Microbiota Transplantation in Pigs.

The intestine of pigs harbors a mass of microorganisms which are essential for intestinal homeostasis and host health. Intestinal microbial disorders induce enteric inflammation and metabolic dysfunction, thereby causing adverse effects on the growth and health of pigs. In the human medicine, fecal microbiota transplantation (FMT), which engrafts the fecal microbiota from a healthy donor into a patient recipient, has shown efficacy in intestinal microbiota restoration. In addition, it has been used widely in therapy for human gastrointestinal diseases, including Clostridium difficile infection, inflammatory bowel diseases, and irritable bowel syndrome. Given that pigs share many similarities with humans, in terms of anatomy, nutritional physiology, and intestinal microbial compositions, FMT may also be used to restore the normal intestinal microbiota of pigs. However, feasible procedures for performing FMT in pigs remains unclear. Here, we summarize a standardized preparation for FMT in pigs by combining the standard methodology for human FMT with pig production. The key issues include the donor selection, fecal material preparation, fecal material transfer, stool bank establishment, and the safety for porcine FMT. Optimal donors should be selected to ensure the efficacy of porcine FMT and reduce the risks of transmitting infectious diseases to recipients during FMT. Preparing for fresh fecal material is highly recommended. Alternatively, frozen fecal suspension can also be prepared as an optimal choice because it is convenient and has similar efficacy. Oral administration of fecal suspension could be an optimal method for porcine fecal material transfer. Furthermore, the dilution ratio of fecal materials and the frequency of fecal material transfer could be adjusted according to practical situations in the pig industry. To meet the potential large-scale requirement in the pig industry, it is important to establish a stool bank to make porcine FMT readily available. Future studies should also focus on providing more robust safety data on FMT to improve the safety and tolerability of the recipient pigs. This standardized preparation for porcine FMT can facilitate the development of microbial targeted therapies and improve the intestinal health of pigs.

Lactobacillus frumenti Facilitates Intestinal Epithelial Barrier Function Maintenance in Early-Weaned Piglets.

Increased intestinal epithelial barrier function damages caused by early weaning stress have adverse effects on swine health and feed utilization efficiency. Probiotics have emerged as the promising antibiotic alternatives used for intestinal barrier function damage prevention. Our previous data showed that Lactobacillus frumenti was identified as a predominant Lactobacillus in the intestinal microbiota of weaned piglets. However, whether the intestinal epithelial barrier function in piglets was regulated by L. frumenti is still unclear. Here, piglets received a PBS vehicle or PBS suspension (2 ml, 108 CFU/ml) containing the L. frumenti by oral gavage once a day during the period of 6-20 days of age prior to early weaning. Our data demonstrated that oral administration of L. frumenti significantly improved the intestinal mucosal integrity and decreased the serum endotoxin and D-lactic acid levels in early-weaned piglets (26 days of age). The intestinal tight junction proteins (including ZO-1, Occludin, and Claudin-1) were significantly up-regulated by L. frumenti administration. The serum immunoglobulin G (IgG) levels, intestinal secretory immunoglobulin A (sIgA) levels, and interferon-γ (IFN-γ) levels were significantly increased by L. frumenti administration. Furthermore, our data revealed that oral administration of L. frumenti significantly increased the relative abundances of health-promoting microbes (including L. frumenti, Lactobacillus gasseri LA39, Parabacteroides distasonis, and Kazachstania telluris) and decreased the relative abundances of opportunistic pathogens (including Desulfovibrio desulfuricans and Candida humilis). Functional alteration of the intestinal bacterial community by L. frumenti administration was characterized by the significantly increased fatty acids and protein metabolism and decreased diseases-associated metabolic pathways. These findings suggest that L. frumenti facilitates intestinal epithelial barrier function maintenance in early-weaned piglets and may be a promising antibiotic alternative used for intestinal epithelial barrier function damage prevention in mammals.