Clostridium difficile is an opportunistic pathogen inhabiting the human gut and is the etiological agent causing C. difficile infections (CDI) currently linked to a dysbiosis of the intestinal microbiota due to antibiotic treatment. The incidence of CDI, which manifestation ranges from mild diarrhea to life-threatening conditions, is increasing not only in compromised subjects, but also in traditionally considered low-risk populations. In addition zoonotic and food transmissions are considered sources for C. difficile acquisition. Several virulence factors are related to CDI, the main ones being the production of toxins A and B involved in the clinical manifestation of the infection. The standard treatment for CDI is antibiotics but the recurrence rate of the disease is very high. This fact has attracted the interest to search for prevention/ therapeutic options to reduce the growth and/or toxicity of C. difficile; among others, probiotics, prebiotics, or a combination of both (synbiotics) constitute a promising approach[1]. Our group has developed a method for the real-time monitoring of the cytotoxicity of C. difficile upon human enterocytes (HT29 and Caco2 lines) using the label-free, impedance-based RTCA xCELLigence equipment (ACEA Bioscience Inc.) and following the morphological changes in the optical microscope LumaScope-600 Series (Etaluma) with time-lapsed imaging capture. Cytotoxic dose- and time-dependent effect of clostridial supernatants upon both cell lines was detected with the RTCA and different toxicological parameters (EC50 and LOAEL) were obtained from the sigmoid-curve fit of data; thus, we have proposed this method as a model for the screening of new anti-CDI agents[2]. Furthermore, this model was applied to search for probiotic candidates able to reduce the toxicity of the pathogen; among the twenty strains tested those belonging to Bifidobacterium longum and B. breve species were the most efficient reducing C. difficile toxicity, the RTCA-normalized cell index values being inversely correlated with the amount of the remnant toxin (A and B) presents in the clostridial supernatant. The strain B. longum IPLA20022 showed the highest anti-toxin activity by RTCA. An image analysis of HT29 monolayers, formed on the surface of 2-well ¿-Slide cover slip (Ibidi GmbH), using the Leica TCS AOBS SP8 X CSLM (Leica Microsystems GmbH) was performed; monolayers treated with C. difficile toxigenic supernatant in the presence/ absence of the strain IPLA20022 showed that this probiotic candidate reduced the cellular rounding (due to the contraction of F-actin microstructure) and diminished the occurrence of apoptotic bodies. Besides, it seems that extracellular factors released by B. longum IPLA20022 are involved in its anti-toxin activity. Finally, using the RTCA technology we have also demonstrated that the combination of specific bifidobacteria with prebiotics (certain fructo-oligosaccharides) is able to reduce the toxicity of C.difficile supernatants.

Trabajo presentado en la 4th Conference on Impedance-Based Cellular Assays (IBCA), celebrada en Edimburgo (Escocia) del 6 al 8 de junio de 2018

Peer reviewed