Impact of Dietary Inputs on Carbapenem Resistance Gene Dynamics and Microbial Safety During Bioconversion of Agri-Food Waste and Anaerobic Digestate by Hermetia illucens Larvae

Background/Objectives: Hermetia illucens larvae can efficiently convert agri-food residues into high-protein biomass for animal feed and nutrient-rich frass for soil amendment. However, the potential spread of carbapenem resistance genes (CRGs), which confer resistance to last-resort carbapenem antibiotics, and Enterobacteriaceae, common carriers of these genes and opportunistic pathogens, raises important safety concerns. This study aimed to assess the influence of different agri-food-based diets on Enterobacteriaceae loads and the CRG occurrence during the bioconversion process. Methods: Four experimental diets were formulated from agri-food residues and anaerobic digestate: Diet 1 (peas and chickpea waste), Diet 2 (peas and wheat waste), Diet 3 (onion and wheat waste), and Diet 4 (wheat waste and digestate). Enterobacteriaceae were quantified by viable counts, while five CRGs (blaKPC, blaNDM, blaOXA-48, blaVIM, and blaGES) were detected and quantified using quantitative PCRs (qPCRs). Analyses were performed on individual substrates, formulated diets, larvae (before and after bioconversion), and frass. Results: Plant-based diets sustained moderate Enterobacteriaceae loads. In contrast, the digestate-based diet led to a significant increase in Enterobacteriaceae in both the frass and mature larvae. CRGs were detected only in legume-based diets: blaVIM and blaGES were found in both mature larvae and frass, while blaOXA-48 and blaKPC were found exclusively in either larvae or frass. No CRGs were detected in onion- or digestate-based diets nor in young larvae or diet inputs. Conclusions: The findings suggest that the diet composition may influence the proliferation of Enterobacteriaceae and the persistence of CRGs. Careful substrate selection and process monitoring are essential to minimize antimicrobial resistance risks in insect-based bioconversion systems.