Bacillus cereus is a common foodborne pathogen. Accidently eating food contaminated by B. cereus will cause vomiting or diarrhea, and even death in severe cases. In the present study, a B. cereus strain was isolated from spoiled rice by streak culture. The pathogenicity and drug resistance of the isolated strain were analyzed by drug sensitivity test and PCR amplification of virulence-associated gene respectively. Cultures of the purified strain were injected intraperitoneally into mice to examine their effects on intestinal immunity-associated factors and gut microbial communities, to provide references for the pathogenic mechanism and medication guidance of these spoilage microorganisms. The results showed that the isolated B. cereus strain was sensitive to norfloxacin, nitrofurantoin, tetracycline, minocycline, ciprofloxacin, spectinomycin, clindamycin, erythrocin, clarithromycin, chloramphenicol, levofloxacin, and vancomycin, but resistant to bactrim, oxacillin and penicillin G. The strain carries seven virulence-associated genes including hblA, hblC, hblD, nheA, nheB, nheC and entFM, which are involved in diarrhea-causing toxins production. After infecting mice, the isolated B. cereus strain was found to cause diarrhea in mice, and the expression levels of immunoglobulins and inflammatory factors in the intestinal mucosae of the challenged mice were significantly up-regulated. Gut microbiome analysis showed that the composition of gut microbial community in mice changed after infection with B. cereus. The abundance of the uncultured_bacterium_f_Muribaculaceae in Bacteroidetes, which is a marker of body health, was significantly decreased. On the other hand, the abundance of uncultured_bacterium_f_Enterobacteriaceae, which is an opportunistic pathogen in Proteobacteria and a marker of dysbacteriosis, was significantly increased and was significantly positively correlated with the concentrations of IgM and IgG. These results showed that the pathogenic B. cereus carrying diarrhea type virulence-associated gene can activate the immune system by altering the composition of gut microbiota upon infection.
Animals ; Mice ; Bacillus cereus/metabolism* ; Food Microbiology ; Immunity, Mucosal ; Diarrhea ; Microbiota ; Enterotoxins/genetics*

Objective To investigate the effect of long intergenic non-coding RNA COX2 (lincRNA-COX2) on apoptosis and polarization of Listeria monocytogenes (Lm)-infected RAW264.7 cells. Methods RAW264.7 cells were cultured and divided into control group (uninfected cells), Lm infection group, negative control of small interfering RNA (si-NC) group, si-NC and Lm infection group, small interfering RNA of lincRNA-COX2 (si-lincRNA-COX2) group, si-lincRNA-COX2 and Lm infection group. RAW264.7 cells were infected with MOI=10 Lm for 6 hours, and then the inhibition efficiency of siRNA transfection was detected by fluorescence microscope and quantitative real-time PCR (qRT-PCR). The expression levels of cleaved-caspase-3(c-caspase-3), caspase-3, B-cell lymphoma-2 (Bcl2), Bcl2 associated X protein (BAX), arginase 1 (Arg1), inducible nitric oxide synthase (iNOS) were detected by Western blot analysis. Results c-caspase-3/caspase-3, BAX/Bcl2 and iNOS were significantly up-regulated, while the level of Arg1 was down-regulated in Lm-infected RAW264.7 cells compared with control group. LincRNA-COX2 knockdown inhibited the increase of protein levels for BAX/Bcl2, c-caspase-3/caspase-3 and iNOS in Lm-infected RAW264.7 cells, while the level of Arg1 in Lm-infected RAW264.7 cells was up-regulated. Conclusion Knockdown of lincRNA-COX2 can inhibit cell apoptosis and suppress the macrophage polarization into M1 type in Lm-infected RAW264.7 cells.
Apoptosis/genetics* ; bcl-2-Associated X Protein/metabolism* ; Caspase 3/metabolism* ; Cyclooxygenase 2/metabolism* ; Listeria monocytogenes/pathogenicity* ; Macrophages/microbiology* ; RNA, Long Noncoding/metabolism* ; RNA, Small Interfering/genetics* ; Animals ; Mice

Marine fungi are important members of the marine microbiome, which have been paid growing attention by scientists in recent years. The secondary metabolites of marine fungi have been reported to contain rich and diverse compounds with novel structures (Chen et al., 2019). Aspergillus terreus, the higher level marine fungus of the Aspergillus genus (family of Trichocomaceae, order of Eurotiales, class of Eurotiomycetes, phylum of Ascomycota), is widely distributed in both sea and land. In our previous study, the coral-derived A. terreus strain C23-3 exhibited potential in producing other biologically active (with antioxidant, acetylcholinesterase inhibition, and anti-inflammatory activity) compounds like arylbutyrolactones, territrems, and isoflavones, and high sensitivity to the chemical regulation of secondary metabolism (Yang et al., 2019, 2020; Nie et al., 2020; Ma et al., 2021). Moreover, we have isolated two different benzaldehydes, including a benzaldehyde with a novel structure, from A. terreus C23-3 which was derived from Pectinia paeonia of Xuwen, Zhanjiang City, Guangdong Province, China.
Acetylcholinesterase/metabolism* ; Animals ; Anthozoa/microbiology* ; Anti-Inflammatory Agents/pharmacology* ; Aspergillus/chemistry* ; Benzaldehydes/pharmacology* ; Mice ; RAW 264.7 Cells ; Signal Transduction

Ma-Mu-Ran Antidiarrheal Capsules (MMRAC) is traditional Chinese medicine that has been used to treat diarrhea caused by acute enteritis (AE) and bacillary dysentery in Xinjiang (China) for many years. However, the potential therapeutic mechanism of MMRAC for AE and its regulatory mechanism on host metabolism is unclear. This study used fecal metabolomics profiling with GC/MS and 16S rRNA gene sequencing analysis to explore the potential regulatory mechanisms of MMRAC on a dextran sulfate sodium salt (DSS)-induced mouse model of AE. Fecal metabolomics-based analyses were performed to detect the differentially expressed metabolites and metabolic pathways. The 16S rRNA gene sequencing analysis was used to assess the altered gut microbes at the genus level and for functional prediction. Moreover, Pearson correlation analysis was used to integrate differentially expressed metabolites and altered bacterial genera. The results revealed that six intestinal bacteria and seven metabolites mediated metabolic disorders (i.e., metabolism of amino acid, carbohydrate, cofactors and vitamins, and lipid) in AE mice. Besides, ten altered microbes mediated the differential expression of eight metabolites and regulated these metabolisms after MMRAC administration. Overall, these findings demonstrate that AE is associated with metabolic disorders and microbial dysbiosis. Further, we present that MMRAC exerts protective effects against AE by improving host metabolism through the intestinal flora.
Animals ; Antidiarrheals/pharmacology* ; Capsules ; Enteritis/genetics* ; Feces/microbiology* ; Genes, rRNA ; Metabolomics ; Mice ; RNA, Ribosomal, 16S/genetics*

Ulcerative Colitis (UC) has been reported to be related to Porphyromonas gingivalis (P. gingivalis). Porphyromonas gingivalis peptidylarginine deiminase (PPAD), a virulence factor released by P. gingivalis, is known to induce inflammatory responses. To explore the pathological relationships between PPAD and UC, we used homologous recombination technology to construct a P. gingivalis strain in which the PPAD gene was deleted (Δppad) and a Δppad strain in which the PPAD gene was restored (comΔppad). C57BL/6 mice were orally gavaged with saline, P. gingivalis, Δppad, or comΔppad twice a week for the entire 40 days (days 0-40), and then, UC was induced by dextran sodium sulfate (DSS) solution for 10 days (days 31-40). P. gingivalis and comΔppad exacerbated DDS-induced colitis, which was determined by assessing the parameters of colon length, disease activity index, and histological activity index, but Δppad failed to exacerbate DDS-induced colitis. Flow cytometry and ELISA revealed that compared with Δppad, P. gingivalis, and comΔppad increased T helper 17 (Th17) cell numbers and interleukin (IL)-17 production but decreased regulatory T cells (Tregs) numbers and IL-10 production in the spleens of mice with UC. We also cocultured P. gingivalis, Δppad, or comΔppad with T lymphocytes in vitro and found that P. gingivalis and comΔppad significantly increased Th17 cell numbers and decreased Treg cell numbers. Immunofluorescence staining of colon tissue paraffin sections also confirmed these results. The results suggested that P. gingivalis exacerbated the severity of UC in part via PPAD.
Animals ; Colitis, Ulcerative/microbiology* ; Mice ; Mice, Inbred C57BL ; Porphyromonas gingivalis/pathogenicity* ; Protein-Arginine Deiminases ; Virulence Factors

To study the interaction between C4b-binding protein (C4BP) and Riemerella anatipestifer (RA), we cloned duck C4BPα, conducted prokaryotic expression and prepared the polyclonal antibody by immunizing mice. Then indirect immunofluorescence assay and dot blotting hybridization assay were used to verify the interaction between C4BP and RA. The full length of duck C4BPα nucleotide sequence was 1 230 bp, with the highest similarity to chicken C4BPα (82.1%). Phylogenetic tree analysis showed that duck C4BPα and chicken C4BPα were on the same phylogenetic tree branch and the genetic evolution relationship between them was the closest. C4BPα was efficiently expressed in Escherichia coli BL21 (DE3). The recombinant proteins existed in intracellular soluble form. The titer of polyclonal antibody was more than 1:10 000 and polyclonal antibodies could specifically recognize the recombinant proteins. The results of indirect immunofluorescence assay and dot blot hybridization assay showed that RA could interact with duck C4BP. The results provide a basis to further reveal the pathogenesis of RA.
Animals ; Cloning, Molecular ; Complement C4b-Binding Protein ; chemistry ; genetics ; metabolism ; Ducks ; classification ; genetics ; microbiology ; Gene Expression Regulation ; Mice ; Phylogeny ; Riemerella ; metabolism

OBJECTIVE: To investigate the characteristics of growth and metabolism and the toxicity of under different conditions. METHODS: We observed the growth of and under routine culture conditions and in different pH and salt concentrations, and compared their activities of sugar fermentation using microbiochemical reaction tubes. Four-week-old nude mice were randomized into infection group (=5), infection group (=5) and control group (=5) for intragastric administration of 0.3 mL suspension the two (5×10 cfu/mL) or 0.3 mL normal saline. Samples of the liver, kidney, intestine, feces and blood were taken for analysis of the distribution and toxicity of by fungal culture and histopathological examination. RESULTS: exhibited logarithmic growth at 8-24 h after inoculation and showed stable growth after 24 h. showed optimal growth within the pH value range of 5-7 with a growth pattern identical to that of . grew better than in media containing 5% and 10% NaCl, and could ferment glucose, sucrose, trehalose and sorbitol. could be isolated from the feces, blood, liver and kidney of infected nude mice, and the liver had the highest fungal load (5.7 log cfu/g). could cause pathological changes in the liver and intestine of the mice, but with a lesser severity as compared with . CONCLUSIONS exhibits optimal growth in mildly acidic or neutral conditions with a high salt tolerance, and can potentially penetrate the intestinal barrier into blood and lead to tissue injuries in hosts with immunosuppression.
Animals ; Candida ; growth & development ; isolation & purification ; Candida albicans ; growth & development ; Candidiasis ; microbiology ; Culture Media ; Mice ; Mice, Nude ; Random Allocation

LysR-type transcriptional regulators are involved in the regulation of numerous cellular metabolic processes in Klebsiella pneumoniae, leading to severe infection. Earlier, we found a novel LysR family gene, named kp05372, in a strain of K. pneumoniae (designated GPKP) isolated from forest musk deer. To study the function of this gene in relation to the biological characteristics of GPKP, we used the suicide plasmid and conjugative transfer methods to construct deletion mutant strain GPKP-Δkp05372; moreover, we also constructed the GPKP-Δkp05372+ complemented strain. The role of this gene was determined by comparing the following characteristics of three strains: growth curves, biofilm formation, drug resistance, stress resistance, median lethal dose (LD₅₀), organ colonization ability, and the histopathology of GPKP. Real-time polymerase chain reaction (RT-PCR) was used to test the expression level of seven genes upstream of kp05372. There was no significant difference in the growth rates when comparing the three bacterial strains, and no significant difference was recorded at different osmotic pressures, temperatures, salt contents, or hydrogen peroxide concentrations. The GPKP-Δkp05372 mutant formed a weak biofilm, and the other two strains formed medium biofilm. The drug resistance of the GPKP-Δkp05372 mutant toward cephalothin, cotrimoxazole, and polymyxin B was changed. The acid tolerance of the deletion strain was stronger than that of the other two strains. The LD₅₀ values of the wild-type and complemented strains were 174-fold and 77-fold higher than that of the GPKP-Δkp05372 mutant, respectively. The colonization ability of the GPKP-Δkp05372 mutant in the heart, liver, spleen, kidney, and intestine was the weakest. The three strains caused different histopathological changes in the liver and lungs. In the GPKP-Δkp05372 mutant, the relative expression levels of kp05374 and kp05379 were increased to 1.32-fold and 1.42-fold, respectively, while the level of kp05378 was decreased by 42%. Overall, the deletion of kp05372 gene leads to changes in the following: drug resistance and acid tolerance; decreases in virulence, biofilm formation, and colonization ability of GPKP; and regulation of the upstream region of adjacent genes.
Animals ; Bacterial Proteins/physiology* ; Biofilms ; Deer/microbiology* ; Drug Resistance, Bacterial ; Female ; Klebsiella Infections/pathology* ; Klebsiella pneumoniae/growth & development* ; Male ; Mice ; Transcription Factors/physiology*

OBJECTIVE: A strain of Aspergillus niger (A. niger), capable of releasing bound phenolic acids from wheat bran, was isolated. This strain was identified by gene sequence identification. The antioxidant and anti-inflammatory capacity of ferulic acid released from wheat bran by this A. niger strain (FA-WB) were evaluated. METHODS: Molecular identification techniques based on PCR analysis of specific genomic sequences were conducted; antioxidant ability was examined using oxygen radical absorbance capacity (ORAC), cellular antioxidant activity (CAA) assays, and erythrocyte hemolysis assays. RAW264.7 cells were used as a model to detect anti-inflammatory activity. RESULTS: The filamentous fungal isolate was identified to be A. niger. ORAC and CAA assay showed that FA-WB had better antioxidant activity than that of the ferulic acid standard. The erythrocyte hemolysis assay results suggested that FA-WB could attenuate AAPH-induced oxidative stress through inhibition of reactive oxy gen species (ROS) generation. FA-WB could significantly restore the AAPH-induced increase in intracellular antioxidant enzyme activities to normal levels as well as inhibit the intracellular malondialdehyde formation. TNF-a, IL-6, and NO levels indicated that FA-WB can inhibit the inflammation induced by lipopolysaccharide (LPS). CONCLUSION Ferulic acid released from wheat bran by a new strain of A. niger had good anti-inflammatory activity and better antioxidant ability than standard ferulic acid.
Animals ; Anti-Inflammatory Agents ; metabolism ; pharmacology ; Antioxidants ; metabolism ; pharmacology ; Aspergillus niger ; genetics ; isolation & purification ; metabolism ; Coumaric Acids ; metabolism ; pharmacology ; DNA, Fungal ; analysis ; Dietary Fiber ; microbiology ; Erythrocytes ; drug effects ; metabolism ; Fermentation ; Hep G2 Cells ; Humans ; Interleukin-6 ; metabolism ; Lipopolysaccharides ; pharmacology ; Mice ; RAW 264.7 Cells ; Sheep ; Tumor Necrosis Factor-alpha ; metabolism

A total of 27 endophytic fungal strains were isolated from Huperzia serrata,which were richly distributed in the stems and leaves while less distributed in roots. The 27 strains were identified by Internal Transcribed Spacer( ITS) r DNA molecular method and one of the strains belongs to Basidiomycota phylum,and other 26 stains belong to 26 species,9 general,6 families,5 orders,3 classes of Ascomycota Phylum. The dominant strains were Colletotrichum genus,belonging to Glomerellaceae family,Glomerellales order,Sordariomycetes class,Ascomycota Phylum,with the percentage of 48. 15%. The inhibitory activities of the crude extracts of 27 endophytic fungal strains against acetylcholinesterase( ACh E) and nitric oxide( NO) production were evaluated by Ellman's method and Griess method,respectively. Crude extracts of four fungi exhibited inhibitory activities against ACh E with an IC50 value of 42. 5-62. 4 mg·L~(-1),and some fungi's crude extracts were found to inhibit nitric oxide( NO) production in lipopolysaccharide( LPS)-activated RAW264. 7 macrophage cells with an IC50 value of 2. 2-51. 3 mg·L~(-1),which indicated that these fungi had potential anti-inflammatory activities.The chemical composition of the Et OAc extract of endophytic fungus HS21 was also analyzed by LCMS-IT-TOF. Seventeen compounds including six polyketides,four diphenyl ether derivatives and seven meroterpenoids were putatively identified.
Acetylcholinesterase ; Animals ; Anti-Inflammatory Agents ; isolation & purification ; pharmacology ; Ascomycota ; chemistry ; classification ; isolation & purification ; Cholinesterase Inhibitors ; isolation & purification ; metabolism ; Endophytes ; classification ; isolation & purification ; Huperzia ; microbiology ; Mice ; RAW 264.7 Cells