• Korean Journal of Microbiology
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• v.54 no.3
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• pp.214-221
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• 2018

Clostridium difficile infection (CDI) is a rapidly emerging infection that may have devastating consequences. Prompt and accurate diagnosis is crucial for management and control. The aim of this study was to determine the incidence of C. difficile associated diarrhea among hospitalized patients, and to compare different diagnostic laboratory methods for detection of toxin producing strains in clinical specimens. The study was conducted at a university hospital in Cairo during the period from May 2013 till June 2015. Subjects were under antibiotic therapy and presented with hospital-acquired diarrhea. Four hundred and sixty-five stool specimens were processed by different microbiological methods. C. difficile was recovered in culture in 51 of stool specimens. Of these, 86.3% to 98% were positive for toxin production by 2 different methods. This study showed that antibiotic intake is the major risk factor for development of hospital-acquired diarrhea. We evaluated different microbiological methods for diagnosis of C. difficile. We recommend the use of toxigenic culture as a gold standard for microbiological diagnosis of C. difficile.

• Food Science of Animal Resources
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• v.40 no.4
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• pp.578-587
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• 2020

Clostridioides (Clostridium) difficile is a Gram (+), anaerobic, spore forming, rod shaped bacterium that can produce toxin. The objective of this study is to reveal the presence of C. difficile in meat products, to analyze the ribotype diversity by PCR and to evaluate the antibiotic susceptibility of isolated strains. The organism was isolated in 22 out of 319 (6.9%) examined meat product samples and 9 out of 22 (40.9%) isolates were identified as RT027 and all isolates had the ability of toxin production. In terms of antibiotic susceptibility, all isolates were susceptive to amoxicillin-clavulanic acid, tetracycline and vancomycin and 21 (95.4%) isolates to metronidazole. On the other hand, imipenem and cefotaxim resistance was observed in all. In conclusion, the results of this comprehensive study conducted in Turkey deduced the presence of C. difficile in different meat products. Therefore, these products can be evaluated as a potential contamination source of C. difficile from animals to humans especially for elders, youngsters, long terms wide spectrum antibiotic used and immuno-suppressed individuals.

• Journal of the Korean Society of Food Science and Nutrition
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• v.41 no.9
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• pp.1326-1330
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• 2012

This study was conducted to develop a loop-mediated isothermal amplification (LAMP) method for the detection of Clostridium difficile. The tested target gene was 16S ribosomal RNA. Five different LAMP primer sets were designed, and LAMP was performed. All primer sets targeting the 16S rRNA gene (BIP, FIP, B3, F3, LF, PF) were determined as positive in tcdA-positive, tcdB-postive ($A^+B^+$) and tcdA-negative, tcdB-negative ($A^-B^-$) Clostridium difficile strains. As the LAMP reaction took less than 80 min and did not require expensive machine such as thermocycler, it can be used as a rapid and simple detection method for foodborne pathogens.

• Journal of Microbiology and Biotechnology
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• v.22 no.1
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• pp.50-57
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• 2012

Phospholipase C-${\gamma}l$ (PLC-${\gamma}l$) expression is associated with cellular transformation. Notably, PLC-${\gamma}$ is up-regulated in colorectal cancer tissue and breast carcinoma. Because exotoxins released by Clostridium botulinum have been shown to induce apoptosis and promote growth arrest in various cancer cell lines, we examined here the potential of Clostridium difficile toxin A to selectively induce apoptosis in cells transformed by PLC-${\gamma}l$ overexpression. We found that PLC-${\gamma}l$-transformed cells, but not vector-transformed (control) cells, were highly sensitive to C. difficile toxin A-induced apoptosis and mitotic inhibition. Moreover, expression of the proapoptotic Bcl2 family member, Bim, and activation of caspase-3 were significantly up-regulated by toxin A in PLC-${\gamma}l$-transformed cells. Toxin A-induced cell rounding and paxillin dephosphorylation were also significantly higher in PLC-${\gamma}l$-transformed cells than in control cells. These findings suggest that C. difficile toxin A may have potential as an anticancer agent against colorectal cancers and breast carcinomas in which PLC-${\gamma}l$ is highly up-regulated.

• Journal of Life Science
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• v.28 no.8
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• pp.885-891
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• 2018

Clostridium difficile (C. difficile) toxin A is known to cause acute gut inflammation in humans and animals by triggering cytoskeletal disorganization in gut epithelial cells. In human colonocytes, toxin A blocks microtubule assembly by directly increasing the enzymatic activity of histone deacetylase-6 (HDAC-6), a tubulin-specific deacetylase, thereby markedly decreasing tubulin acetylation, which is essential for microtubule assembly. Microtubule assembly dysfunction-associated alterations (i.e., toxin A-exposed gut epithelial cells) are believed to trigger barrier dysfunction and gut inflammation downstream. We recently showed that potassium acetate blocked toxin A-induced microtubule disassembly by inhibiting HDAC-6. Herein, we tested whether acetic acid (AA), another small acetyl residue-containing agent, could block toxin A-induced tubulin deacetylation and subsequent microtubule assembly. Our results revealed that AA treatment increased tubulin acetylation and enhanced microtubule assembly in an HT29 human colonocyte cell line. AA also clearly increased tubulin acetylation in murine colonic explants. Interestingly, the AA treatment also alleviated toxin A-induced tubulin deacetylation and microtubule disassembly, and MTT assays revealed that AA reduced toxin A-induced cell toxicity. Collectively, these results suggest that AA can block the ability of toxin A to cause microtubule disassembly-triggered cytoskeletal disorganization by blocking toxin A-mediated deacetylation of tubulin.

• Journal of Life Science
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• v.28 no.9
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• pp.1016-1021
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• 2018

Clostridium difficile toxin A causes pseudomembranous colitis. The pathogenesis of toxin A-induced colonic inflammation includes toxin A-dependent epithelial cell apoptosis, resulting in the loss of barrier function provided by epithelial cells against luminal pathogens. Toxin A-dependent epithelial cell apoptosis has been linked to toxin A-induced production of reaction oxygen species and subsequent p38MAPK activation; $p21^{CIP1/WAF1}$ upregulation-dependent cell cycle arrest; cytoskeletal disaggregation; and/or the induction of Fas ligand on epithelial cells. However, the molecular mechanisms underlying toxin A-induced apoptosis remain poorly understood. This study tested whether toxin A could block the Wnt signaling pathway, which is involved in gut epithelial cell proliferation, differentiation and antiapoptotic progression. Toxin A treatment of nontransformed human colonocytes (NCM460) rapidly reduced ${\beta}$-catenin protein, an essential component of the Wnt signaling pathway. Exposure of mouse ileum to toxin A also significantly reduced ${\beta}$-catenin protein levels. MG132 inhibition of proteasome-dependent protein degradation resulted in the recovery of toxin A-mediated reduction of ${\beta}$-catenin, indicating that toxin A may activate intracellular processes, such as $GSK3{\beta}$, to promote degradation of ${\beta}$-catenin. Immunoblot analysis showed that toxin A increased active phosphorylation of $GSK3{\beta}$. Because the Wnt signaling pathway is essential for gut epithelial cell proliferation and anti-apoptotic processes, our results suggest that toxin A-mediated inhibition of the Wnt signaling pathway may be required for maximal toxin A-induced apoptosis of gut epithelial cells.

• Journal of Microbiology and Biotechnology
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• v.22 no.2
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• pp.170-175
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• 2012

Clostridium difficile toxin A glucosylates Rho family proteins, resulting in actin filament disaggregation and cell rounding in cultured colonocytes. Given that the cellular toxicity of toxin A is dependent on its receptor binding and subsequent entry into the cell, we herein sought to identify additional colonocyte proteins that might bind to toxin A following its internalization. Our results revealed that toxin A interacted with ERK1 and ERK2 in two human colonocyte cell lines (NCM460 and HT29). A GST-pulldown assay also showed that toxin A can directly bind to ERK1 and ERK2. In NCM460 cells exposed to PMA (an ERK1/2 activator), the phosphorylation of ERK1/2 did not affect the interaction between toxin A and ERK1/2. However, an in vitro kinase assay showed that the direct binding of toxin A to ERK1 or ERK2 inhibited their kinase activities. These results suggest a new molecular mechanism for the cellular toxicity seen in cells exposed to toxin A.

• Journal of Microbiology and Biotechnology
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• v.29 no.1
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• pp.30-36
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• 2019

Numerous studies have reported that enteric neurons involved in controlling neurotransmitter secretion and motility in the gut critically contribute to the progression of gut inflammation. Clostridium difficile toxins, which cause severe colonic inflammation, are also known to affect enteric neurons. Our previous study showed that C. difficile toxin A directly induces neural cell toxicities, such as viability loss and apoptosis. In the current study, we attempted to identify a potent inhibitor of toxin A-induced neural cell toxicity that may aid in managing toxin A-induced gut inflammation. In our recent study, we found that the Korea dung beetle-derived antimicrobial peptide CopA3 completely blocked neural cell apoptosis caused by okadaic acid or 6-OHDA. Here, we examined whether the antimicrobial peptide CopA3 inhibited toxin A-induced neural cell damage. In neuroblastoma SH-SY5Y cells, CopA3 treatment protected against both apoptosis and viability loss caused by toxin A. CopA3 also completely inhibited activation of the pro-apoptotic factor, caspase-3. Additionally, CopA3 rescued toxin A-induced downregulation of neural cell proliferation. However, CopA3 had no effect on signaling through ROS/p38 $MAPK/p27^{kip1}$, suggesting that CopA3 inhibits toxin A-induced neural cell toxicity independent of this well-characterized toxin A pathway. Our data further suggest that ability of CopA3 to rescue toxin A-induced neural cell damage may also ameliorate the gut inflammation caused by toxin A.

• Pediatric Gastroenterology, Hepatology & Nutrition
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• v.17 no.4
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• pp.232-238
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• 2014

Purpose: This study investigated the clinical presentations of symptomatic Clostridium difficile infection (CDI) in children. Methods: We reviewed the medical records of 43 children aged <20 years who showed either positive C. difficile culture or C. difficile toxin test results between June 2010 and April 2014. Results: Of the 43 patients (mean age 6.7 years), 22 were boys. Sixteen patients (37.2%) showed both positive C. difficile culture and toxin test results. Seventeen out of 43 children (39.5%) had preexisting gastrointestinal diseases, and 26 children had other medical conditions that were risk factors for CDI. Twenty-eight children had a history of antibiotic treatment for >3 days, and the most frequently prescribed antibiotic was amoxicillin-clavulanate (35.7%). Twenty-eight patients were diagnosed with CDI despite taking probiotic supplements, most commonly Lactobacillus acidophilus (53.6%). The most common symptom was diarrhea (72.1%) at the time CDI was diagnosed. C. difficile was eradicated in 11 patients (25.6%) after treatment with oral metronidazole for 10-14 days, and in the two patients (4.6%) who required two courses of oral metronidazole. Sixteen patients (37.2%) showed clinical improvement without any treatment. Conclusion: This study showed the various clinical characteristics of CDI in children and that preexisting clinical conditions favored the development of CDI. In addition, CDI was found to occur in a number of patients even after probiotic prophylaxis given in conjunction with antibiotic therapy.

• Food Science of Animal Resources
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• v.41 no.5
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• pp.883-893
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• 2021

Clostridium difficile present in feces of food animals may contaminate their meats and act as a potential source of C. difficile infection (CDI) to humans. C. difficile resistance to antibiotics, its production of toxins and spores play major roles in the pathogenesis of CDI. This is the first study to evaluate C. difficile prevalence in retail raw animal meats, its antibiotics susceptibilities and toxigenic activities in Al-Jouf, Saudi Arabia. Totally, 240 meat samples were tested. C. difficile was identified by standard microbiological and biochemical methods. Vitek-2 compact system confirmed C. difficile isolates were 15/240 (6.3%). Toxins A/B were not detected by Xpect C. difficile toxin A/B tests. Although all isolates were susceptible to vancomycin and metronidazole, variable degrees of reduced susceptibilities to moxifloxacin, clindamycin or tetracycline antibiotics were detected by Epsilon tests. C. difficile strains with reduced susceptibility to antibiotics should be investigated. Variability between the worldwide reported C. difficile contamination levels could be due to absence of a gold standard procedure for its isolation. Establishment of a unified testing algorithm for C. difficile detection in food products is definitely essential to evaluate the inter-regional variation in its prevalence on national and international levels. Proper use of antimicrobials during animal husbandry is crucial to control the selective drug pressure on C. difficile strains associated with food animals. Investigating the protective or pathogenic potential of non-toxigenic C. difficile strains and the possibility of gene transfer from certain toxigenic/ antibiotics-resistant to non-toxigenic/antibiotics-sensitive strains, respectively, should be worthy of attention.