Clostridium difficile is an important zoonotic intestinal pathogen, which is widely present in humans and a variety of animals. The ST11 type C. difficile is one of the most widespread and harmful subtypes in the world. As a large country in pig farming, China lacks efficient methods for detecting C. difficile of porcine origin, leaving hidden dangers for the prevention and control of C. difficile. The aim of this study was to develop a specific and sensitive double-antibody sandwich ELISA for the epidemiological investigation of ST11 type C. difficile of porcine origin. Firstly, a 97 kDa receptor binding domain (RBD) was expressed in a prokaryotic host and purified. A hybridoma cell line AE2D3 capable of stably secreting monoclonal antibody targeting the RBD was screened, and the antibody subtype was determined to be IgG2b (κ). Secondly, a double antibody sandwich ELISA method was developed, where the monoclonal antibody targeting the RBD was used as a detection antibody, and the rabbit polyclonal antibody was used as a capture antibody. The chessboard method was used to determine the matching concentration of the capture antibody and the detection antibody, the antigen coating conditions, the blocking conditions, the incubation conditions for detection antibody and samples to be tested, as well as the reaction conditions of HRP-conjugated and reaction conditions of TMB chromogenic solution. The negative cutoff OD₄₅₀ was 0.152, and no cross-reaction with 13 strains of non-ST11 type C. difficile was found. The minimum detection concentration of RBD was 8.83 ng/mL. This specific and sensitive double-antibody sandwich ELISA provides a reliable serological detection method for epidemiological investigation of the ST11 type C. difficile in pig industry.
Animals ; Antibodies, Monoclonal ; Bacterial Proteins/genetics* ; Bacterial Toxins ; Clostridioides difficile ; Enzyme-Linked Immunosorbent Assay ; Hybridomas ; Swine

Animals ; Anthrax ; microbiology ; Antigens, Bacterial ; genetics ; Bacillus anthracis ; genetics ; Bacterial Toxins ; genetics ; Gene Expression Regulation, Bacterial ; Humans ; Receptors, Peptide ; genetics

Bacteria are often infected by large numbers of phages, and host bacteria have evolved diverse molecular strategies in the race with phages, with abortive infection (Abi) being one of them. The toxin-antitoxin system (TA) is expressed in response to bacterial stress, mediating hypometabolism and even dormancy, as well as directly reducing the formation of offspring phages. In addition, some of the toxins' sequences and structures are highly homologous to Cas, and phages even encode antitoxin analogs to block the activity of the corresponding toxins. This suggests that the failure of phage infection due to bacterial death in abortive infections is highly compatible with TA function, whereas TA may be one of the main resistance and defense forces for phage infestation of the host. This review summarized the TA systems involved in phage abortive infections based on classification and function. Moreover, TA systems with abortive functions and future use in antibiotic development and disease treatment were predicted. This will facilitate the understanding of bacterial-phage interactions as well as phage therapy and related synthetic biology research.
Anti-Bacterial Agents ; Antitoxins/chemistry* ; Bacteria/genetics* ; Bacterial Proteins/chemistry* ; Bacterial Toxins/genetics* ; Bacteriophages/genetics* ; Toxin-Antitoxin Systems/genetics*

To investigate the occurrence and distribution of serotype, specific virulence genes, and pulse field gel electrophoresis (PFGE) patterns in Vibrio parahaemolyticus isolates from Jeonnam, Korea, we tested 87 strains which were identified with V. parahaemolyticus from diarrheal episode patients in 2005. In this study, 16 different O:K serotype combinations of V. parahaemolyticus were determined. The distributions of O and K serotypes were O4:K68 (51.72%), O1:K70 (18.39%), O3:K6 (5.74%), O1:K68 (4.60%) and O3:K57 (4.60%) respectively. Serotype O4:K68 was the regional dominant specific serotype of V. parahaemolyticus in Sinan of Jeonnam, Korea. For the detection of thermostable direct hemolysin (tdh) and TDH-related hemolysin (trh) gene of V. parahaemolyticus, PCR was performed. The tdh gene was detected in all of the V. parahaemolyticus isolates from diarrheal patients, but trh gene was not detected. Analysis of PFGE patterns of 30 V. parahaemolyticus isolates showed 3 groups and 20 types. Among 14 O4:K68 serotypes which were isolated in Sinan, PFGE patterns of 12 strains were closely related (100%), but 2 strains were related by 58.3% and 45.4%, respectively. Also two strains of O1:K4 serotype in Gurye and two strains of O3:K6 serotype in Yeosu were closely related (100%), respectively. Although serotypes (O1:K4, O1:K70, O3:K6 and O4:K68) were different, PFGE patterns were related for more than 80.9%. Therefore, the epidemiological surveillance of V. parahaemolyticus is required by PFGE typing scheme as a further diagnostic tool.
Bacterial Toxins ; Electrophoresis ; Hemolysin Proteins ; Humans ; Korea ; Polymerase Chain Reaction ; Serotyping ; Vibrio ; Vibrio parahaemolyticus

PURPOSE: The cardiopulmonary exercise test (CPET) is an important clinical tool for evaluating exercise capacity and is frequently used to evaluate chronic conditions including congenital heart disease. However, data on the normal CPET values for Korean children and adolescents are lacking. The aim of this study was to provide reference data for CPET variables in children and adolescents. METHODS: From August 2006 to April 2009, 76 healthy children and adolescents underwent the CPET performed using the modified Bruce protocol. Here, we performed a medical record review to obtain data regarding patient' demographics, medical history, and clinical status. RESULTS: The peak oxygen uptake (VO2Peak) and metabolic equivalent (METMax) were higher in boys than girls. The respiratory minute volume (VE)/CO2 production (VCO2) slope did not significantly differ between boys and girls. The cardiopulmonary exercise test data did not significantly differ between the boys and girls in younger age group (age, 10 to 14 years). However, in older age group (age, 15 to 19 years), the boys had higher VO2Peak and METMax values and lower VE/VCO2 values than the girls. CONCLUSION: This study provides reference data for CPET variables in case of children and adolescents and will make it easier to use the CPET for clinical decision-making.
Adolescent ; Bacterial Toxins ; Child ; Demography ; Exercise Test ; Heart Diseases ; Humans ; Medical Records ; Metabolic Equivalent ; Oxygen

This study was aimed to investigate the effect of clostridium difficile toxin A (Tcd A) on proliferation of K562 cells and its mechanism. The proliferative activity of K562 cells exposed to Tcd A was tested by MTT assay; cell cycle distribution and mitochondrial membrane potential were analyzed by flow cytometry; the protein expression of cytochrome C and DNA fragmentation were observed by immunohistochemistry staining and agarose gel electrophoresis respectively. The results indicated that Tcd A inhibited proliferation of K562 cells in a time-and concentration-dependent manner. Cells were arrested at G(0)/G(1) phase. Peak of apoptosis appeared. The protein expression of cytochrome C increased as compared with control group (p < 0.05). Agarose gel electrophoresis of DNA from K562 treated with Tcd A revealed a "ladder" pattern. It is concluded that clostridium difficile toxin A can inhibit proliferation and induce apoptosis of K562 cells. The mechanism may be in relation to decrease of mitochondrial membrane potential and the release of cytochrome C from mitochondria matrix.
Apoptosis ; drug effects ; Bacterial Toxins ; pharmacology ; Cell Proliferation ; drug effects ; Enterotoxins ; pharmacology ; Humans ; K562 Cells

The purpose of the study is to establish a colorimetric method of HEC toxin hemolysis test for diagnosis of paroxysmal nocturnal hemoglobinuria (PNH). RBCs from normal persons and patients with PNH and non-PNH anemia were treated with HEC toxin secreted by Aeromonas hydrophila J-1 strain and the absorbance at 630 nm was measured to quantitate the extent of hemolysis. The results demonstrated that the RBCs from PNH patients showed resistance to the toxin hemolysis, which was in accord with the percentages of CD59(-) cells, while the RBCs from normal persons and non-PNH anemic patients were nearly totally lysed. It is concluded that the method can be considered as a simple, specific and reliable method for the diagnosis of PNH.
Bacterial Toxins ; toxicity ; Colorimetry ; Flow Cytometry ; Hemoglobinuria, Paroxysmal ; diagnosis ; Hemolysis ; Humans

Objective: To establish and optimize PCR methods for the gene encoding of Clostridium perfringens β₂ toxin (cpb₂) and atypical-cpb₂ (aty-cpb₂), analyze the epidemiological characteristics and genetic polymorphism of the cpb₂ of Clostridium perfringens in 9 Chinese areas from 2016 to 2021. Methods: The cpb₂ of 188 Clostridium perfringens strains were examined by PCR; the cpb₂ sequences were acquired by whole-genome sequencing to analyze the genetic polymorphism. Using Mega 11 and the Makeblastdb tool, a phylogenetic tree, and cpb₂-library based on 110 strains carrying the cpb₂ were produced. Using the Blastn technique, a comparison was made to discover sequence similarity between consensus-cpb₂ (con-cpb₂) and aty-cpb₂. Results: The specificity of PCR assay for the cpb₂ and aty-cpb₂ was verified. The PCR results for cpb₂ amplification were highly consistent with the whole-genome sequencing approach (Kappa=0.946, P<0.001). A total of 107 strains from nine regions in China carried cpb₂, 94 types A strains carried aty-cpb₂, 6 types A strains carried con-cpb₂, and 7 types F strains carried aty-cpb₂. The nucleotide sequence similarity between the two coding genes was 68.97%-70.97%, and the similarity between the same coding genes was 98.00%-100.00%. Conclusions: In this study, a specific PCR method for cpb₂ toxin was developed, and the previous PCR method for detecting aty-cpb₂ was improved. aty-cpb₂ is the primary gene encoding of β₂ toxin. There is a significant nucleotide sequence variance between the various cpb₂ genotypes.
Humans ; Clostridium perfringens/genetics* ; Clostridium Infections ; Bacterial Toxins/genetics* ; Phylogeny ; Polymerase Chain Reaction ; Polymorphism, Genetic

Objective: Comparative analyses of wild-type Clostridioides difficile 630 (Cd630) strain and pathogenicity locus (PaLoc) knockout mutant (ΔPaLoc) by using RNA-seq technology. Analysis of differential expression of Cd630 wild-type strain and ΔPaLoc mutant strain and measurement of its cellular virulence changes. Lay the foundation for the construction of an toxin-attenuated vaccine strain against Clostridioides difficile. Methods: Analysis of Cd630 and ΔPaLoc mutant strains using high-throughput sequencing (RNA-seq). Clustering differentially expressed genes and screening differentially expressed genes by DESeq software. Further analysis of differential genes using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment. Finally, cytotoxicity assays of ΔPaLoc and Cd630 strains were performed in the African monkey kidney epithelial cell (Vero) and the human colonic cell (Caco-2) lines. Results: The transcriptome data showed that the ΔPaLoc mutant toxin genes tcdA and tcdB were not transcribed. Compared to the wild-type strain, CD630_36010, CD630_020910,CD630_02080 and cel genes upregulated 17.92,11.40,8.93 and 7.55 fold, respectively. Whereas the hom2 (high serine dehydrogenase), the CD630_15810 (spore-forming protein), CD630_23230 (zinc-binding dehydrogenase) and CD630_23240 (galactitol 1-phosphate 5-dehydrogenase) genes were down-regulated by 0.06, 0.075, 0.133 and 0.183 fold, respectively. The GO and KEGG enrichment analyses showed that the differentially transcribed genes in ΔPaLoc were enriched in the density-sensing system, ABC transport system, two-component system, phosphotransferase (PTS) system, and sugar metabolism pathway, as well as vancomycin resistance-related pathways. Cytotoxicity assays showed that the ΔPaLoc mutant strain lost its virulence to Vero and Caco-2 cells compared to the wild-type Cd630 strain. Conclusion: Transcriptional sequencing analysis of the Cd630 and ΔPaLoc mutant strains showed that the toxin genes were not transcribed. Those other differential genes could provide a reference for further studies on the physiological and biochemical properties of the ΔPaLoc mutant strain. Cytotoxicity assays confirmed that the ΔPaLoc mutant lost virulence to Vero and Caco-2 cells, thus laying the foundation for constructing an toxin-attenuated vaccine strain against C. difficile.
Bacterial Proteins/metabolism* ; Bacterial Toxins/metabolism* ; Caco-2 Cells ; Clostridioides ; Clostridioides difficile/genetics* ; Humans ; Oxidoreductases/metabolism* ; Transcriptome ; Vaccines, Attenuated

Animals ; Bacterial Toxins ; chemistry ; pharmacology ; therapeutic use ; Humans ; Neoplasms ; therapy ; Neurotoxins ; chemistry ; pharmacology ; therapeutic use ; Proteins ; chemistry ; pharmacology ; therapeutic use ; Toxins, Biological ; chemistry ; pharmacology ; therapeutic use