Detection of intracellular toxins A and B in epidemic genotypes of Clostridiodes difficile
DOI:
https://doi.org/10.17533/10.17533/udea.hm.v12n1a02Keywords:
Clostridioides difficile, toxins, intracellular, NAP1/RT027Abstract
Introduction: Clostridioides difficile is the world’s leading cause of hospital-acquired diarrhea due to antibiotic use, with toxins A and B (TcdA and TcdB) as the main virulence factors. In recent years, there has been an emergence of strains with increased virulence, characterized as genotype NAP1/RT027, which are responsible for epidemic outbreaks. These strains exhibit a higher toxin secretion and high fluoroquinolone resistance. The first report of epidemic outbreaks due to these strains in Latin America was described in Costa Rica, where an autochthonous genotype was also described and classified as NAPCR1/RT019. This genotype showed increased virulence but no toxin oversecretion.
Methods: Cell lysates from clinical isolates of NAP1/ RT027, NAPCR1/RT019, and NAP4/RT014 genotypes were obtained and intracellular TcdA and TcdB were detected by Western Blot (WB). In addition, a cell culture cytotoxicity assay was performed to evaluate the level and biological activity of TcdB in those lysates. This was performed at different times on the in vitro bacterial growth.
Results: TcdA was detected in the cell lysates of all study genotypes and TcdB was detected only in NAP1/RT027 by WB. The highest TcdA and TcdB signal was detected in NAP1/RT027 at 24 and 48 hours of growth. On cell culture, the predominant cytotoxic activity was due to TcdB produced by NAP1/RT027, followed by NAPCR1/RT019 and a lower activity in NAP4/RT014.
Conclusions: The detection of a higher amount and activity of intracellular toxins in the epidemic strain NAP1/RT027 correlates with previous reports of toxin overproduction by this genotype. It was proved that a strain with increased virulence, such as NAPCR1/RT019 does not overproduce toxins. This also correlates with reports of extracellular toxin detection. This study presents important information about methodologies for toxin detection on C. difficile lysates and contributes to the discussion on the differences in toxin production according to this pathogen’s genotypes.
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