Bacillus cereus belongs to Gram-positive bacteria, which is widely distributed in nature and shows certain pathogenicity. Different B. cereus strains carry different subsets of virulence factors, which directly determine the difference in their pathogenicity. It is therefore important to study the distribution of virulence factors and the biological activity of specific toxins for precise prevention and control of B. cereus infection. In this study, the hemolysin BL triayl was expressed, purified, and characterized. The results showed that the bovine pathogenic B. cereus hemolysin BL could be expressed and purified in the prokaryotic expression system, and the bovine pathogenic B. cereus hemolysin BL showed hemolysis, cytotoxicity, good immunogenicity and certain immune protection in mice. In this study, the recombinant expression of hemolysin BL triayl was achieved, and the biological activity of hemolysin BL of bovine pathogenic ceroid spore was investigated. This study may facilitate further investigating the pathogenic mechanism of B. cereus hemolysin BL and developing a detection method for bovine pathogenic B. cereus disease.
Cattle ; Animals ; Mice ; Bacterial Proteins/metabolism* ; Bacillus cereus/metabolism* ; Hemolysin Proteins/metabolism* ; Virulence Factors/metabolism* ; Enterotoxins/metabolism*

OBJECTIVES: To explore the molecular characteristics of Staphylococcus aureus (S. aureus) in children, and to compare the molecular characteristics of different types of strains (infection and colonization strains) so as to reveal pathogenic molecular markers of S. aureus. METHODS: A cross-sectional study design was used to conduct nasopharyngeal swab sampling from healthy children in the community and clinical samples from infected children in the hospital. Whole genome sequencing was used to detect antibiotic resistance genes and virulence genes. A random forest method to used to screen pathogenic markers. RESULTS: A total of 512 S. aureus strains were detected, including 272 infection strains and 240 colonization strains. For virulence genes, the carrying rates of enterotoxin genes (seb and sep), extracellular enzyme coding genes (splA, splB, splE and edinC), leukocytotoxin genes (lukD, lukE, lukF-PV and lukS-PV) and epidermal exfoliating genes (eta and etb) in infection strains were higher than those in colonization strains. But the carrying rates of enterotoxin genes (sec, sec3, seg, seh, sei, sel, sem, sen, seo and seu) were lower in infection strains than in colonization strains (P<0.05). For antibiotic resistance genes, the carrying rates of lnuA, lnuG, aadD, tetK and dfrG were significantly higher in infection strains than in colonization strains (P<0.05). The accuracy of cross-validation of the random forest model for screening pathogenic markers of S. aureus before and after screening was 69% and 68%, respectively, and the area under the curve was 0.75 and 0.70, respectively. The random forest model finally screened out 16 pathogenic markers (sem, etb, splE, sep, ser, mecA, lnuA, sea, blaZ, cat(pC233), blaTEm-1A, aph(3')-III, ermB, ermA, ant(9)-Ia and ant(6)-Ia). The top five variables in the variable importance ranking were sem (OR=0.40), etb (OR=3.95), splE (OR=1.68), sep (OR=3.97), and ser (OR=1.68). CONCLUSIONS The random forest model can screen out pathogenic markers of S. aureus and exhibits a superior predictive performance, providing genetic evidence for tracing highly pathogenic S. aureus and conducting precise targeted interventions.
Child ; Humans ; Staphylococcus aureus/genetics* ; Cross-Sectional Studies ; Enterotoxins/genetics* ; Staphylococcal Infections ; Whole Genome Sequencing

Objective: We analyze the characteristics of Clostridioides difficile (C. difficile) infection among diarrhea patients in Kunming from 2018 to 2020 and provide evidence for follow-up surveillance and prevention. Methods: A total of 388 fecal samples of diarrhea patients from four sentinel hospitals in Yunnan Province from 2018 to 2020 were collected. Real-time quantitative PCR was used to detect the fecal toxin genes of C. difficile. The positive fecal samples isolated the bacteria, and isolates were identified by mass spectrometry. The genomic DNA of the strains was extracted for multi-locus sequence typing (MLST). The fecal toxin, strain isolation, and clinical patient characteristics, including co-infection with other pathogens, were analyzed. Results: Among the 388 fecal samples, 47 samples with positive reference genes of C. difficile were positive, with a total positive rate of 12.11%. There were 4 (8.51%) non-toxigenic and 43 (91.49%) toxigenic ones. A total of 18 strains C. difficile were isolated from 47 positive specimens, and the isolation rate of positive specimens was 38.30%. Among them, 14 strains were positive for tcdA, tcdB, tcdC, tcdR, and tcdE. All 18 strains of C. difficile were negative for binary toxins. The MLST results showed 10 sequence types (ST), including 5 strains of ST37, accounting for 27.78%; 2 strains of ST129, ST3, ST54, and ST2, respectively; and 1 strain of ST35, ST532, ST48, ST27, and ST39, respectively. Fecal toxin gene positive (tcdB+) results were statistically associated with the patient's age group and with or without fever before the visit; positive isolates were only statistically associated with the patient's age group. In addition, some C. difficile patients have co-infection with other diarrhea-related viruses. Conclusions: The infection of C. difficile in diarrhea patients in Kunming is mostly toxigenic strains, and the high diversity of strains was identified using the MLST method. Therefore, the surveillance and prevention of C. difficile should be strengthened.
Humans ; Bacterial Toxins/genetics* ; Enterotoxins/genetics* ; Clostridioides difficile/genetics* ; Multilocus Sequence Typing ; Coinfection ; Bacterial Proteins/genetics* ; China/epidemiology* ; Clostridium Infections/epidemiology* ; Diarrhea/microbiology*

Objective: The docking and superantigen activity sites of staphylococcal enterotoxin-like W (SElW) and T cell receptor (TCR) were predicted, and its SElW was cloned, expressed and purified. Methods: AlphaFold was used to predict the 3D structure of SElW protein monomers, and the protein models were evaluated with the help of the SAVES online server from ERRAT, Ramachandran plot, and Verify_3D. The ZDOCK server simulates the docking conformation of SElW and TCR, and the amino acid sequences of SElW and other serotype enterotoxins were aligned. The primers were designed to amplify selw, and the fragment was recombined into the pMD18-T vector and sequenced. Then recombinant plasmid pMD18-T was digested with BamHⅠand Hind Ⅲ. The target fragment was recombined into the expression plasmid pET-28a(+). After identification of the recombinant plasmid, the protein expression was induced by isopropyl-beta-D- thiogalactopyranoside. The SElW expressed in the supernatant was purified by affinity chromatography and quantified by the BCA method. Results: The predicted three-dimensional structure showed that the SElW protein was composed of two domains, the amino-terminal and the carboxy-terminal. The amino-terminal domain was composed of 3 α-helices and 6 β-sheets, and the carboxy-terminal domain included 2 α-helices and 7 antiparallel β-sheets composition. The overall quality factor score of the SElW protein model was 98.08, with 93.24% of the amino acids having a Verify_3D score ≥0.2 and no amino acids located in disallowed regions. The docking conformation with the highest score (1 521.328) was selected as the analysis object, and the 19 hydrogen bonds between the corresponding amino acid residues of SElW and TCR were analyzed by PyMOL. Combined with sequence alignment and the published data, this study predicted and found five important superantigen active sites, namely Y18, N19, W55, C88, and C98. The highly purified soluble recombinant protein SElW was obtained with cloning, expression, and protein purification. Conclusions: The study found five superantigen active sites in SElW protein that need special attention and successfully constructed and expressed the SElW protein, which laid the foundation for further exploration of the immune recognition mechanism of SElW.
Humans ; Enterotoxins/genetics* ; Superantigens/genetics* ; Catalytic Domain ; Selenoprotein W/metabolism* ; Receptors, Antigen, T-Cell

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*

PURPOSE: Osteitis refers to the development of new bone formation and remodeling of bone in chronic rhinosinusitis (CRS) patients; it is typically associated with eosinophilia, nasal polyps (NPs), and recalcitrant CRS. However, the roles of ossification in CRS with or without NPs remain unclear due to the lack of appropriate animal models. Thus, it is necessary to have a suitable animal model for greater advances in the understanding of CRS pathogenesis.METHODS: BALB/c mice were administered ovalbumin (OVA) and staphylococcal enterotoxin B (SEB). The numbers of osteoclasts and osteoblasts and bony changes were assessed. Micro computed tomography (micro-CT) scans were conducted to measure bone thickness. Immunofluorescence, immunohistochemistry, and quantitative polymerase chain reaction were performed to evaluate runt-related transcription factor 2 (RUNX2), osteonectin, interleukin (IL)-13, and RUNX2 downstream gene expression. Gene set enrichment analysis was performed in mucosal tissues from control and CRS patients. The effect of resveratrol was evaluated in terms of osteogenesis in a murine eosinophilic CRS NP model.RESULTS: The histopathologic changes showed markedly thickened bones with significant increase in osteoblast numbers in OVA/SEB-treated mice compared to the phosphate-buffered saline-treated mice. The structural changes in bone on micro-CT were consistent with the histopathological features. The expression of RUNX2 and IL-13 was increased by the administration of OVA/SEB and showed a positive correlation. RUNX2 expression mainly co-localized with osteoblasts. Bioinformatic analysis using human CRS transcriptome revealed that IL-13-induced bony changes via RUNX2. Treatment with resveratrol, a candidate drug against osteitis, diminished the expression of IL-13 and RUNX2, and the number of osteoblasts in OVA/SEB-treated mice.CONCLUSIONS: In the present study, we found the histopathological and radiographic evidence of osteogenesis using a previously established murine eosinophilic CRS NP model. This animal model could provide new insights into the pathophysiology of neo-osteogenesis and provide a basis for developing new therapeutics.
Animals ; Computational Biology ; Core Binding Factor Alpha 1 Subunit ; Enterotoxins ; Eosinophilia ; Eosinophils ; Fluorescent Antibody Technique ; Gene Expression ; Humans ; Immunohistochemistry ; Interleukin-13 ; Interleukins ; Mice ; Models, Animal ; Mucous Membrane ; Nasal Polyps ; Nose ; Osteitis ; Osteoblasts ; Osteoclasts ; Osteogenesis ; Osteonectin ; Ovalbumin ; Polymerase Chain Reaction ; Sinusitis ; Transcription Factors ; Transcriptome

PURPOSE: Staphylococcal enterotoxin B (SEB) has been well-documented to induce liver injury. miRNA-222-3p (miR-222-3p) was implicated in SEB-induced lung injury and several liver injuries. This study aimed to explore the role of miR-222-3p in SEB-induced liver injury. MATERIALS AND METHODS: Expression of miR-222-3p and suppressors of cytokine signaling 1 (SOCS1) was detected using real-time quantitative PCR and western blot. Liver injury was determined by levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and inflammatory cytokines, numbers of infiltrating mononuclear cells using AST/ALT assay kit, enzyme-linked immunosorbent assay (ELISA), and hematoxylin-eosin staining, respectively. Target binding between miR-222-3p and SOCS1 was predicted on targetScan software, and confirmed by luciferase reporter assay. RESULTS: SEB induced liver injury in D-galactosamine (D-gal)-sensitized mice, as demonstrated by increased serum levels of AST and ALT, elevated release of interferon-gamma (INF-γ), tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and IL-2, and promoted infiltrating immune cells into liver. Expression of miR-222-3p was dramatically upregulated, and SOCS1 was downregulated in SEB-induced liver injury both in mice and splenocytes. Moreover, miR-222-3p knockout (KO) mice exhibited alleviated liver injury accompanied with SOCS1 upregulation. Besides, splenocytes under SEB challenge released less INF-γ, TNF-α, IL-6, and IL-2 during miR-222-3p knockdown. Mechanically, SOCS1 was targeted and downregulated by miR-222-3p. Upregulation of SOCS1 attenuated INF-γ, TNF-α, IL-6, and IL-2 release in SEB-induced splenocytes; downregulation of SOCS1 could block the suppressive role of miR-222-3p knockdown in SEB-induced splenocytes. CONCLUSION: Inhibition of miR-222-3p relieves SEB-induced liver inflammatory injury by upregulating SOCS1, thereby providing the first evidence of miR-222-3p in SEB-induced liver injury.
Alanine Transaminase ; Animals ; Aspartate Aminotransferases ; Blotting, Western ; Cytokines ; Down-Regulation ; Enterotoxins ; Enzyme-Linked Immunosorbent Assay ; Interferon-gamma ; Interleukin-2 ; Interleukin-6 ; Liver ; Luciferases ; Lung Injury ; Mice ; Polymerase Chain Reaction ; Tumor Necrosis Factor-alpha ; Up-Regulation

Claudin proteins are the most crucial components of tight junctions, and play an essential role in maintaining cell polarity, regulating cell permeability and the intercellular ion. In recent years, many studies have shown that abnormality of claudins expression is implicated in the tumor progression. The expression correlates with tumor prognosis and can serve as a biomarker of prognosis and potential therapeutic targets. This review summarizes the current knowledge regarding claudin dysregulation in cancer and highlights the progress in claudin-based treatments.
Claudins ; therapeutic use ; Enterotoxins ; Humans ; Neoplasms ; drug therapy ; Tight Junctions

The recent emergence of Staphylococcus schleiferi in dogs with otitis externa or skin and soft tissue infections has become a significant zoonotic issues. In the current study, we investigated 1) the carriage rates of S. schleiferi among major staphylococci in healthy dogs and dogs with otitis externa, 2) antibiotic susceptibility profiles of S. schleiferi, particularly methicillin resistance (MR), and 3) virulence factors associated with skin and soft tissue infections such as ability to form biofilm, resistance to cationic antimicrobial peptides (CAMPs), and carriage of staphylococcal enterotoxin genes. Among the 21 S. schleiferi isolates, 5 isolates (24%) were determined to be methicillin-resistant (MRSS). Staphylococcal cassette chromosome mec (SCCmec) typing revealed the presence of SCCmec type V in 4 MRSS isolates and type VII in one MRSS. Higher levels of antibiotic resistance, especially multidrug resistance, were observed in MRSS isolates compared to the methicillin-susceptible S. schleiferi (MSSS) isolates. In addition, MRSS isolates exhibited enhanced ability to form biofilm under static condition and all the 5 MRSS isolates carried three or more enterotoxin genes. However, there were no significant differences in resistance to CAMPs between MRSS and MSSS isolates. These findings suggest that coagulase-negative S. schleiferi is becoming more prevalent in canine otitis externa cases. Our results also highlight the presence of multidrug-resistant MRSS isolates with enhanced biofilm production and carriage of multiple enterotoxins.
Animals ; Antimicrobial Cationic Peptides ; Biofilms ; Dogs ; Drug Resistance, Microbial ; Drug Resistance, Multiple ; Enterotoxins ; Methicillin Resistance ; Otitis Externa ; Otitis ; Skin ; Soft Tissue Infections ; Staphylococcus ; Virulence Factors ; Virulence

As an important zoonotic pathogen, Staphylococcus aureus has led to serious mastitis and endometritis in infected dairy cows. In this study, a total of 164 strains of S. aureus were isolated from dairy cows in Xinjiang Province, China, and subjected to assays to determine drug susceptibility and biofilm (BF) formation ability. Enterotoxin-related genes were detected, and the transcription levels of genes related to BF formation were determined by using reverse transcription-quantitative polymerase chain reaction. Moreover, the pathogenicity of isolates with different BF formation abilities was determined by measuring their hemolysis activity, half lethal dose (LD₅₀) and organ bacterial load. The results showed that 86.0% of S. aureus isolates could form BF. Among them, 42.1% of the strains had weak BF formation ability, and most strains with a strong BF formation ability were ica gene carriers. The S. aureus isolates displayed multidrug resistance and their drug resistance was positively correlated with their BF formation ability. Moreover, 96.3% of the S. aureus isolates carried enterotoxin genes. Among them, the detection rates of the novel enterotoxin genes were higher than those of conventional enterotoxin genes. Furthermore, isolates with a strong BF formation ability had higher LD50 but lower hemolysis ability and organ bacterial load than those of the isolates with weak or no BF ability. However, isolates without BF ability produced more severe pathological changes than those of isolates with strong BF formation ability. These findings suggest that higher BF ability and presence of novel enterotoxin genes are important characteristics of S. aureus isolates from dairy cows in Xinjiang Province, China, and such isolates may pose potential threats to food safety.
Bacterial Load ; Biofilms ; China ; Drug Resistance ; Drug Resistance, Microbial ; Drug Resistance, Multiple ; Endometritis ; Enterotoxins ; Female ; Food Safety ; Hemolysis ; Lethal Dose 50 ; Mastitis ; Polymerase Chain Reaction ; Staphylococcus aureus ; Staphylococcus ; Virulence