Antimicrobial kinetics of a blend of bioactive compounds and its role in maintaining epithelial integrity during a Clostridium perfringens challenge on a chicken intestinal cell line

Necrotic enteritis is one of the most common diseases in poultry production worldwide, leading to sub-optimal performance. Aim of this study was to investigate the antimicrobial kinetics of a blend of bioactive compounds against Clostridium perfringens, the causative agent of necrotic enteritis. Moreover, the role of this blend in counteracting a bacterial challenge on a chicken intestinal cell line was assessed by measuring epithelial integrity. ATCC® 13124 Clostridium perfringens (CP) strain was cultured at 37°C in Reinforced Clostridial Medium in an anaerobic environment. For the time-kill assay, CP at 105 CFU/ml was grown without treatments (CTR group) or in presence of either a high dose (HD) or a low dose (LD) of the bioactive compounds blend (botanicals and fatty acids). CP was plated on TSA blood agar every hour, from inoculation (0 hours) for the first 8 hours, and after 24 hours in order to count viable bacteria over time. For the cell culture experiment, chicken intestinal epithelial cells (cIEC) were seeded on 0.4 µm transwell inserts and allowed to grow in specific expansion media at 37°C and 5% CO2, until complete polarization. Then, cells were challenged with CP at 105 CFU/ml without treatments (CTR+) or with either bacitracin at 2 ppm, the HD or the LD blend (n=6). The CTR- group was used as not-challenged control. The integrity of the epithelial layer was assessed by measuring trans-epithelial electrical resistance (TEER) at 0 hours, 1.5 hours, and 3 hours post-challenge. Data were analyzed with 2-way ANOVA and differences considered significant at p<0.05. In the time-kill assay, both the HD and LD blend allowed a reduction of CP growth compared to the control right after the inoculation and the HD blend completely killed bacteria at 0 hours (p<0.0001). The LD blend significantly decreased CP growth in a more gradual manner and after 4 hours totally killed the microorganism, with no difference from HD blend. During the bacterial challenge on cIEC, CP strongly reduced the TEER at 1.5 hours (-65% than initial value) and even more at 3 hours (-95% than 0 hours). Despite the challenge, the LD group was able to protect the cells preventing TEER drop by 40% and 45% at 1.5 and 3 hours, respectively, compared with the challenged control (p<0.0001). Moreover, bacitracin and HD groups showed a TEER comparable to the not-challenged control at both time-points. The investigated blend of bioactive compounds demonstrated a bactericidal activity against Clostridium perfringens and protected epithelial cells during a bacterial in vitro challenge in a dose-dependent manner.

Key Words: Clostridium perfringens; feed additives; timekill assay; TEER; bioactive compounds

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Discover More: Giovagnoni et al. (2022). PSA 111th annual meeting, Poult. Sci., 102, 195