Bacteriophage-mediated decolonization of Enterobacteriaceae in a novel Galleria mellonella gut colonization model

Purpose Galleria mellonella larvae have emerged as an invertebrate model for studying bacterial pathogenesis and novel therapeutic options due to ethical concerns associated with the use of mammalian models such as mice. The benefits of using G. mellonella larvae include a less complex microbiome in the gut, making it suitable for gut colonization studies. The intestinal colonization of Klebsiella pneumoniae (Kp) and Escherichia coli (Ec), two of the most antibiotic-resistant bacteria on the World Health Organization’s (WHO) priority list, plays a key role in the spread of antibiotic resistance. Bacteriophage therapy is emerging as a promising alternative for antibiotic-resistant bacteria due to its ability to specifically target and infect bacterial hosts, making it suitable for gut decontamination. This study aimed to establish a novel Enterobacteriaceae G. mellonella larvae gut colonization model and compare the efficacy of conventional antibiotic treatment with a one-time phage cocktail in decolonizing the gut. Approach Larvae were force-fed with different concentrations of bacterial doses of K. pneumoniae and E. coli at 0 h, 24 h, and 48 h, followed by survival monitoring at 24 h intervals. After 48 h and 120 h of the last force feed, the colony forming unit (CFU) count in the gut was evaluated. After successful colonization, larvae were one-time force-fed with either a 107 PFU/larvae bacteriophage cocktail or with ciprofloxacin 4 mg/L or meropenem 2 mg/L. After 24 h post phage feeding, CFU counts were determined. Main findings Three bacterial doses of 106 CFU/larvae led to a stable gut colonization in the larvae gut regardless of the K. pneumoniae and E. coli strains. Bacteriophage force-feeding reduced bacterial colonization by 4 log10 CFU/larvae whereas antibiotic treatment led to a 2 log10 CFU/larvae reduction compared to the control. The novel alternative G. mellonella model for gut colonization studies can be used for proof-of-concept studies, reducing or even obviating the number of follow-up experiments in vertebrate models. ### Competing Interest Statement The authors have declared no competing interest.