Impact of Bacillus licheniformis from yaks following antibiotic therapy in mouse model.

Gut microorganism (GM) is an integral component of the host microbiome and health system. Abuse of antibiotics disrupts the equilibrium of the microbiome, affecting environmental pathogens and host-associated bacteria alike. However, relatively little research on Bacillus licheniformis alleviates the adverse effects of antibiotics. To test the effect of B. licheniformis as a probiotic supplement against the effects of antibiotics, cefalexin was applied, and the recovery from cefalexin-induced jejunal community disorder and intestinal barrier damage was investigated by pathology, real-time PCR (RT-PCR), and high-throughput sequencing (HTS). The result showed that A group (antibiotic treatment) significantly reduced body weight and decreased the length of jejunal intestinal villi and the villi to crypt (V/C) value, which also caused structural damage to the jejunal mucosa. Meanwhile, antibiotic treatment suppressed the mRNA expression of tight junction proteins ZO-1, claudin, occludin, and Ki67 and elevated MUC2 expression more than the other Groups (P < 0.05 and P < 0.01). However, T group (B. licheniformis supplements after antibiotic treatment) restored the expression of the above genes, and there was no statistically significant difference compared to the control group (P > 0.05). Moreover, the antibiotic treatment increased the relative abundance of 4 bacterial phyla affiliated with 16 bacterial genera in the jejunum community, including the dominant Firmicutes, Proteobacteria, and Cyanobacteria in the jejunum. B. licheniformis supplements after antibiotic treatment reduced the relative abundance of Bacteroidetes and Proteobacteria and increased the relative abundance of Firmicutes, Epsilonbacteraeota, Lactobacillus, and Candidatus Stoquefichus. This study uses mimic real-world exposure scenarios by considering the concentration and duration of exposure relevant to environmental antibiotic contamination levels. We described the post-antibiotic treatment with B. licheniformis could restore intestinal microbiome disorders and repair the intestinal barrier. KEY POINTS: • B. licheniformis post-antibiotics restore gut balance, repair barrier, and aid health • Antibiotics harm the gut barrier, alter structure, and raise disease risk • Long-term antibiotics affect the gut and increase disease susceptibility.

Effect of Lysinibacillus isolated from environment on probiotic properties and gut microbiota in mice.

Soil microorganisms (SM) are primarily involved in organism degradation, plant nitrogen nutrient immobilization, host microorganisms and oxidation. However, research on the effect of soil-derived Lysinibacillus on the intestinal microbiota spatial disparity of mice is lacking. To test the probiotic properties of Lysinibacillus and the spatial disparity on mice intestinal microorganisms, hemolysis test, molecular phylogenetic analysis, antibiotic sensitivity testing, serum biochemical assays and 16S rRNA profiling were applied. The results showed that Lysinibacillus (LZS1 and LZS2) was resistant to two common antibiotics, Tetracyclines and Rifampin, and sensitive to other antibiotics among the 12 antibiotics tested and negative for hemolysis. In addition, the body weight of group L (treatment of Lysinibacillus, 1.0 × 108 CFU/d for 21days) mice was significantly greater than that of the control group; serum biochemical tests showed that the TG and UREA were significantly lower in group L. The spatial disparity of intestinal microorganisms in mice was significant, treatment of Lysinibacillus (1.0 × 108 CFU/d for 21days) reduced the intestinal microbial diversity and decreased the richness of Proteobacteria, Cyanobacteria and Bacteroidetes in mice. Furthermore, Lysinibacillus treatment enhanced Lactobacillus and Lachnospiraceae richness and significantly reduced 6 bacterial genera in jejunum community, reduced 8 bacterial genera, but increased bacteria at the 4 genera level in cecum microorganisms. In conclusion, this study demonstrated spatial disparity of intestinal microorganisms in mice and probiotic potential of Lysinibacillus isolated from soil.

Bacillus licheniformis reverses the environmental ceftriaxone sodium-induced gut microbial dysbiosis and intestinal inflammation in mice.

Antibiotics used as a common clinical treatment have saved many lives. Widespread use of antibiotic therapy has been known to disrupt the balance of pathogenic bacteria, host-associated microorganisms and environment. However, our understanding of Bacillus licheniformis for health benefits and ability to restore the ceftriaxone sodium-induced gut microbial dysbiosis is severely limited. We used Caco-2 cell, H&E (hematoxylin-eosin staining), RT-PCR and 16S rRNA sequencing techniques to investigate the influence of Bacillus licheniformis on gut microbial dysbiosis and inflammation following ceftriaxone sodium treatment. The results showed that treatment of ceftriaxone sodium in 7 days suppressed the expression of Nf-κB pathway mRNA levels, which caused cytoplasmic vacuolization in intestinal tissues, afterward, the administration of Bacillus licheniformis could effectively restore intestinal morphology and inflammation levels. Moreover, the ceftriaxone sodium treatment entirely affected the intestinal microbial ecology, leading to a decrease in microbial abundance. Firmicutes, Proteobacteria, and Epsilonbacteraeota were the most predominant phyla in each of the four groups. Specifically, the MA group (ceftriaxone sodium treatment) resulted in a significant decrease in the relative abundance of 2 bacterial phyla and 20 bacterial genera compared to the administration of Bacillus licheniformis after ceftriaxone sodium treatment. The supplementation of Bacillus licheniformis could increase the growth of Firmicutes and Lactobacillus and encourage the construction of a more mature and stable microbiome. Furthermore, Bacillus licheniformis could restore the intestinal microbiome disorders and inflammation levels following ceftriaxone sodium treatment.

Baicalin inhibits apoptosis and enhances chondrocyte proliferation in thiram-induced tibial dyschondroplasia in chickens by regulating Bcl-2/Caspase-9 and Sox-9/Collagen-II expressions.

Avian tibial dyschondroplasia (TD) is a skeletal disease affecting fast growing chickens, resulting in non-mineralized avascular cartilage. This metabolic disorder is characterized by lameness and reduced growth performance causing economic losses. The aim of this study was to investigate the protective effects of baicalin against TD caused by thiram exposure. A total of two hundred and forty (n = 240) one day-old broiler chickens were uniformly and randomly allocated into three different groups (n = 80) viz. control, TD, and baicalin groups. All chickens received standard feed, however, to induce TD, the TD and baicalin groups received thiram (tetramethylthiuram disulfide) at a rate of 50 mg/kg feed from days 4-7. The thiram induction in TD and baicalin groups resulted in lameness, high mortality, and enlarged growth-plate, poor production performance, reduction in ALP, GSH-Px, SOD, and T-AOC levels, and increased AST and ALT, and MDA levels. Furthermore, histopathological results showed less vascularization, and mRNA and protein expression levels of Sox-9, Col-II, and Bcl-2 showed significant downward trend, while caspase-9 displayed significant up-regulation in TD-affected chickens. After the TD induction, the baicalin group was orally administered with baicalin at a rate of 200 mg/kg from days 8-18. Baicalin administration increased the vascularization, and chondrocytes with intact nuclei, alleviated lameness, decreased GP size, increased productive capacity, and restored the liver antioxidant enzymes and serum biochemical levels. Furthermore, baicalin significantly up-regulated the gene and protein expressions of Sox-9, Col-II, and Bcl-2, and significantly down-regulated the expression of caspase-9 (p < 0.05). Therefore, the obtained results suggest that baicalin could be a possible choice in thiram toxicity alleviation by regulating apoptosis and chondrocyte proliferation in thiram-induced tibial dyschondroplasia.

Prevalence and molecular characterization of Cryptosporidium spp. in yaks (Bos grunniens) in Naqu, China.

The intestinal protozoan specie, Cryptosporidium causes serious diarrheal syndrome in humans and animals worldwide. However, limited knowledge is known about the infection caused by this specie in yaks in Naqu. About 950 serum and 150 fecal samples were collected and assayed by using commercial ELISA kits and nPCR detection methods to find the prevalence and molecular characterization of Cryptosporidium spp. in yaks. Results found that 103 out of 950 (10.8%) serums were uncovered against C. parvum antibodies. In different regions, the prevalence of C. parvum in yaks were in a range from 9.1% to 16.7%, with obvious difference among the three areas (P < 0.001). In male and female yaks, the prevalence of C. parvum was found to be 7.2% and 13.3% respectively (P < 0.001); and a significant difference (P < 0.001) with a range of 9.8%-18.2% was observed among different age groups. Out of 150 fecal samples, only 2 (1.3%) positive samples were detected via nPCR. The positive samples were sequenced and identified to be C. bovis. The two isolates were clustered to cattle and yak clade separately. Our results highlight the prevalence and epidemiological status of Cryptosporidium spp. in yaks which may contribute towards the prevention and control of this zoonotic disease in Naqu, China.

Comparative analysis of microbial community structure in the ponds with different aquaculture model and fish by high-throughput sequencing.

The pond has a complex microbial ecosystem, including microorganisms in water and sediment, which plays an important role in the health of fish and water quality. The microbial community structure in the ponds can be easily affected by many factors. However, not much is known about the role of different aquaculture model and fish on the microbial community structure in ponds. The purpose of the study was to investigate the microbial diversity and composition of the ponds with different aquaculture model and fish by high-throughput sequencing. A total of 3835072 valid sequences were achieved from 60 samples. Additionally, 2064 and 1917 core OTUs were observed in water and sediment samples, respectively. Our results suggested that sediment samples have a higher abundance and diversity of microbial community than water samples. In all the samples, the four most dominant phyla were Proteobacteria, Cyanobacteria, Actinomycetes and Bacteroides. At the genus level, hgcI_clade and CL500-29_marine_group were the dominant bacteria shared by the water samples and sediment samples. In addition, more bacteria related to eutrophication were found in the group of BF, BC and HSB, which suggested that these ponds may have been eutrophicated. In conclusion, the present study revealed the differences in the structure and diversity of microbial communities in ponds with different aquaculture model and fish. Furthermore, changes in typical bacteria of the ponds contribute to detect water quality and prevent water eutrophication.

Experimental Study and Application of Controlled Low-Strength Materials in Trench Backfilling in Suqian City, China.

When backfilling narrow spaces, controlled low-strength materials (CLSM) can be used to achieve an effective backfilling effect. The pipeline engineering in Yahnghe Avenue of Suqian, China, provides a favorable on-site condition for the use of CLSM. However, no guidance exists for the determination of the material mixture ratio of CLSM for this geological condition. Laboratory tests were performed to investigate the basic physical parameters of excavated soil and the optimal mixture ratio of CLSM. Results indicate that the sand and silt account for 29.76% and 57.23% of the weight of excavated soil, respectively. As the water content increases (from 40% to 50%), the flowability of the CLSM approximately shows a linear increase (slumps values from 154.3 mm to 269.75 mm for 9% cement content), while its compressive strength shows a linear decreasing trend (from 875.3 KPa to 468.3 KPa after curing for 28 days); as the cement content increases (from 6% to 12%), the flowability approximately shows a linear decreasing trend (from 238.8 mm to 178.5 mm for 45% water content), while the compressive strength shows a linear increasing trend (from 391.6 KPa to 987.6 KPa after curing for 28 days). By establishing the relationship between compressive strength/flowability and the water-cement ratio, the optimal material ratio is determined to be 9% cement content and 40-43% water content. The engineering application results indicate that the use of CLSM can achieve efficient and high-quality backfilling effects for pipeline trenches. The findings of this research may provide a reference for the application of CLSM in fields with similar geological conditions.

Gut microbiota and metabolic modulation by supplementation of polysaccharide-producing Bacillus licheniformis from Tibetan Yaks: A comprehensive multi-omics analysis.

Gut microbiota and their metabolic processes depend on the intricate interplay of gut microbiota and their metabolic processes. Bacillus licheniformis, a beneficial food supplement, has shown promising effects on stabilizing gut microbiota and metabolites. However, the precise mechanisms underlying these effects remain elusive. In this study, we investigated the impact of polysaccharide-producing B. licheniformis as a dietary supplement on the gut microbiome and metabolites through a combination of scanning electron microscopy (SEM), histological analysis, high-throughput sequencing (HTS), and metabolomics. Our findings revealed that the B. licheniformis-treated group exhibited significantly increased jejunal goblet cells. Moreover, gut microbial diversity was lower in the treatment group as compared to the control, accompanied by noteworthy shifts in the abundance of specific bacterial taxa. Enrichment of Firmicutes, Lachnospiraceae, and Clostridiales_bacterium contrasted with reduced levels of Campylobacterota, Proteobacteria, Parasutterella, and Helicobacter. Notably, the treatment group showed significant weight gain after 33 days, emphasizing the polysaccharide's impact on host metabolism. Delving into gut metabolomics, we discovered significant alterations in metabolites. Nine metabolites, including olprinone, pyruvic acid, and 2-methyl-3-oxopropanoate, were upregulated, while eleven, including defoslimod and voclosporin were down-regulated, shedding light on phenylpropanoid biosynthesis, tricarboxylic acid cycle (TCA cycle), and the glucagon signaling pathway. This comprehensive multi-omics analysis offers compelling insights into the potential of B. licheniformis as a dietary polysaccharide supplement for gut health and host metabolism, promising significant implications for gut-related issues.

Lactobacillus plantarum modulate gut microbiota and intestinal immunity in cyclophosphamide-treated mice model.

Gut microbiota (GM) contributes to the production of immune-regulatory molecules and cytokines. However, our understanding regarding intricate relationship between Lactobacillus plantarum and GM on regulation of immune function remained limited. To investigate the effect of Lactobacillus plantarum on an immunosuppressed mouse model, we employed cyclophosphamide treatment and conducted various analysis including H&E (hematoxylin-eosin staining), immunohistochemistry, 16S rRNA gene sequencing, and RT-PCR. Our results demonstrated that the administration of Lactobacillus plantarum had significant immunoenhancing effects in the immune-suppressed mice, as evidenced by the restoration of functional expression of specific immune markers in the spleen and an increase in the number of goblet cells in intestine (P < 0.05). Microbial taxonomic analysis revealed alterations in the gut microbiota composition, characterized by a decrease in the richness of Firmicutes and an increase in the proportion of Verrucomicrobia and Actinobacteria following cyclophosphamide treatment. Furthermore, cyclophosphamide treatment significantly suppressed the mRNA expression of inflammatory cytokines (P < 0.05), which were subsequently restored after administration of Lactobacillus plantarum. These observations provide valuable insights into the complex interplay between probiotics, gut microbiota, and immune system functioning.

Music-based interventions in the feeding environment on the gut microbiota of mice.

Gut microbiota is established to be associated with the diversity of gastrointestinal conditions, but information on the variation associated with music and gut microbes is limited. Current study revealed the impacts of music intervention during feeding on the growth performance and gut microbes of mice by using clinical symptoms and 16S rRNA sequencing techniques. The results showed that feeding mice with music had a significant increase in body weight after the 25th day. The Firmicutes and Proteobacteria were the most dominant phylum in the gut microbiota. Also, the relative abundance of the dominant bacteria was variable after musical intervention. In contrast to the control group, a significant decrease in alpha diversity analysis of gut bacterial microorganisms and Metastats analysis showed a significant increase in the relative abundance of 5 genera and one phylum after the music intervention. Moreover, the musical intervention during feeding caused modifications in the gut microbial composition of mice, as evidenced by an increase in the level of Firmicutes and Lactobacillus, while decreases the richness of pathogenic bacteria, e.g. Proteobacteria, Cyanobacteria and Muribaculaceae, etc. In summary, music intervention increased body weight and enhanced the abundance of beneficial bacteria by reducing the prevalence of pathogenic bacteria in gut microbiota of mice.

Detection of Culex tritaeniorhynchus Giles and Novel Recombinant Strain of Lumpy Skin Disease Virus Causes High Mortality in Yaks.

Lumpy skin disease virus (LSDV) is capable of causing transboundary diseases characterized by fever, nodules on the skin, mucous membranes, and inner organs. The disease may cause emaciation with the enlargement of lymph nodes and sometimes death. It has had endemic importance in various parts of Asia in recent years, causing substantial economic losses to the cattle industry. The current study reported a suspected LSDV infection (based on signs and symptoms) from a mixed farm of yak and cattle in Sichuan Province, China. The clinical samples were found positive for LSDV using qPCR and ELISA, while LSDV DNA was detected in Culex tritaeniorhynchus Giles. The complete genome sequence of China/LSDV/SiC/2021 was determined by Next-generation sequencing. It was found that China/LSDV/SiC/2021 is highly homologous to the novel vaccine-related recombinant LSDV currently emerging in China and countries surrounding China. Phylogenetic tree analysis revealed that the novel vaccine-associated recombinant LSDV formed a unique dendrograms topology between field and vaccine-associated strains. China/LSDV/SiC/2021 was found to be a novel recombinant strain, with at least 18 recombination events via field viruses identified in the genome sequence. These results suggest that recombinant LSDV can cause high mortality in yaks, and its transmission might be due to the Culex tritaeniorhynchus Giles, which acts as a mechanical vector.

Probiotic Potential of Bacillus licheniformis and Bacillus pumilus Isolated from Tibetan Yaks, China.

Yak (Bos grunniens) inhabit an oxygen-deficient environment at the altitude of 3000 m on the Tibetan Plateau, with a distinctive gut micro-ecosystem. This study evaluated the probiotic potential and physiological property of Bacillus licheniformis and Bacillus pumilus isolated from the gut of yaks. Four strains, two Bacillus licheniformis (named D1 and D2) and two Bacillus pumilus (named X1 and X2), were isolated and identified by 16S rRNA sequencing. All strains had potential antibacterial ability against three indicator pathogens: Escherichia coli C83902, Staphylococcus aureus BNCC186335, and Salmonella enteritidis NTNC13349. The antioxidant activity test showed that D2 sample showed the highest antioxidant activity. Furthermore, all four strains had a higher hydrophobicity, auto-aggregation, acid tolerance, bile tolerance, and antibiotic sensitivity, which all contribute to their survival in the gastrointestinal tract and clinical utility. The animal experimentation (40 KM mice, equally divided into five groups of eight mice each) showed that the strain supplementation not only increased daily weight gain and reduced feed conversion ratio, but also increased the length of the jejunum villi and the value of the V/C (Villi/Crypt). In conclusion, this is the first study demonstrated the probiotic potential of Bacillus licheniformis and Bacillus pumilus isolated from yaks, providing a theoretical basis for the clinical application and development of new feed additives.

Effects of Short-Chain Fatty Acid Modulation on Potentially Diarrhea-Causing Pathogens in Yaks Through Metagenomic Sequencing.

Short-chain fatty acids (SCFA) are principal nutrient substrates of intestinal epithelial cells that regulate the epithelial barrier in yaks. Until now, metagenomics sequencing has not been reported in diarrheal yaks. Scarce information is available regarding the levels of fecal SCFA and diarrhea in yaks. So, our study aims to identify the potential pathogens that cause the emerging diarrhea and explore the potential relationship of short-chain fatty acids in this issue. We estimated diarrhea rate in yaks after collecting an equal number of fecal samples from affected animals. Metagenomics sequencing and quantitative analysis of SCFA were performed, which revealed 15%-25% and 5%-10% prevalence of diarrhea in yak's calves and adults, respectively. Violin box plot also showed a higher degree of dispersion in gene abundance distribution of diarrheal yaks, as compared to normal yaks. We found 366,163 significant differential abundance genes in diarrheal yaks, with 141,305 upregulated and 224,858 downregulated genes compared with normal yaks via DESeq analysis. Metagenomics binning analysis indicated the higher significance of bin 33 (Bacteroidales) (p < 0.05) in diarrheal animals, while bin 10 (p < 0.0001), bin 30 (Clostridiales) (p < 0.05), bin 51 (Lactobacillales) (p < 0.05), bin 8 (Lachnospiraceae) (p < 0.05), and bin 47 (Bacteria) (p < 0.05) were significantly higher in normal yaks. At different levels, a significant difference in phylum (n = 4), class (n = 8), oder (n = 8), family (n = 16), genus (n = 17), and species (n = 30) was noticed, respectively. Compared with healthy yaks, acetic acid (p < 0.01), propionic acid (p < 0.01), butyric acid (p < 0.01), isobutyric acid (p < 0.01), isovaleric acid (p < 0.05), and caproic acid (p < 0.01) were all observed significantly at a lower rate in diarrheal yaks. In conclusion, besides the increased Staphylococcus aureus, Babesia ovata, Anaplasma phagocytophilum, Bacteroides fluxus, viruses, Klebsiella pneumonia, and inflammation-related bacteria, the decrease of SCFA caused by the imbalance of intestinal microbiota was potentially observed in diarrheal yaks.

Probiotic Properties of Bacillus proteolyticus Isolated From Tibetan Yaks, China.

Yaks (Bos grunniens) live primarily in high-altitude hypoxic conditions and have a unique intestinal micro-ecosystem, remarkable adaptability, and strong climatic resistance. Accumulating evidence revealed the importance of probiotics in host metabolism, gut microbiota, growth performance, and health. The goal of this study was to screen out probiotics with excellent probiotic potential for clinical application. In this study, four strains of Bacillus, i.e., Bacillus proteolyticus (named Z1 and Z2), Bacillus amyloliquefaciens (named J), and Bacillus subtilis (named K), were isolated and identified. Afterward, their probiotic potential was evaluated. Antioxidant activity tests revealed that Z1 had the highest DPPH and hydroxyl radical scavenging activity, whereas Z2 had higher reducing power and inhibited lipid peroxidation. Additionally, the antibacterial testing revealed that all strains were antagonistic to three indicator pathogens, Escherichia coli C83902, Staphylococcus aureus BNCC186335, and Salmonella enteritidis NTNC13349. These isolates also had a higher hydrophobicity, autoaggregation, and acid and bile tolerance, all of which helped to survive and keep dangerous bacteria out of the host intestine. Importantly, all strains could be considered safe in terms of antibiotic susceptibility and lack of hemolysis. In conclusion, this is the first study to show that B. proteolyticus and B. amyloliquefaciens isolated from yaks have probiotic potential, providing a better foundation for future clinical use.

The Complete Genome of Probiotic Lactobacillus sakei Derived from Plateau Yak Feces.

Probiotic bacteria are receiving increased attention due to the potential benefits to their hosts. Plateau yaks have resistance against diseases and stress, which is potentially related to their inner probiotics. To uncover the potential functional genes of yak probiotics, we sequenced the whole genome of Lactobacillus sakei (L. sakei). The results showed that the genome length of L. sakei was 1.99 Mbp, with 1943 protein coding genes (21 rRNA, 65 tRNA, and 1 tmRNA). There were three plasmids found in this bacteria, with 88 protein coding genes. EggNOG annotation uncovered that the L. sakei genes were found to belong to J (translation, ribosomal structure, and biogenesis), L (replication, recombination, and repair), G (carbohydrate transport and metabolism), and K (transcription). GO annotation showed that most of the L. sakei genes were related to cellular processes, metabolic processes, biological regulation, localization, response to stimulus, and organization or biogenesis of cellular components. CAZy annotation found that there were 123 CAZys in the L. sakei genome, with glycosyl transferases and glycoside hydrolases. Our results revealed the genome characteristics of L. sakei, which may give insight into the future employment of this probiotic bacterium for its functional benefits.