Synergistic and off-target effects of bacteriocins in a simplified human intestinal microbiome: implications for <i>Clostridioides difficile</i> infection control

<i>Clostridioides difficile</i> is a major cause of nosocomial diarrhea. As current antibiotic treatment failures and recurrence of infections are highly frequent, alternative strategies are needed for the treatment of this disease. This study explores the use of bacteriocins, specifically lacticin 3147 and pediocin PA-1, which have reported inhibitory activity against <i>C. difficile</i>. We engineered <i>Lactococcus lactis</i> strains to produce these bacteriocins individually or in combination, aiming to enhance their activity against <i>C. difficile</i>. Our results show that lacticin 3147 and pediocin PA-1 display synergy, resulting in higher anti-<i>C. difficile</i> activity. We then evaluated the effects of these <i>L. lactis</i> strains in a Simplified Human Intestinal Microbiome (SIHUMI-C) model, a bacterial consortium of eight diverse human gut species that includes <i>C. difficile</i>. After introducing the bacteriocin-producing <i>L. lactis</i> strains into SIHUMI-C, samples were collected over 24 hours, and the genome copies of each species were assessed using qPCR. Contrary to expectations, the combined bacteriocins increased <i>C. difficile</i> levels in the consortium despite showing synergy against <i>C. difficile</i> in agar-based screening. This can be rationally explained by antagonistic inter-species interactions within SIHUMI-C, providing new insights into how broad-spectrum antimicrobials might fail to control targeted species in complex gut microbial communities. These findings highlight the need to mitigate off-target effects in complex gut microbiomes when developing bacteriocin-based therapies with potential clinical implications for infectious disease treatment.