In Vitro Elimination of Highly Multidrug-Resistant Bacteria by the Lactic Acid Bacterial Drug Candidate ILP100

Introduction: Multidrug resistance (MDR) has been identified in wound bacterial isolates from Ukrainian war victims treated in Ukraine and across Europe. ILP100, a drug candidate for the treatment of skin wounds, is composed of a Limosilactobacillus reuteri expressing human chemokine CXCL12. In this study, the antimicrobial effects of ILP100 were tested on MDR bacteria isolated from wounds of Ukrainian war victims. Methods: ILP100 was co-cultured with one of the wound pathogens (Pseudomonas aeruginosa, Acinetobacter baumannii, Enterobacter cloacae, Klebsiella pneumoniae, Proteus mirabilis, Staphylococcus aureus; 12 non-MDR and 12 MDR isolates) in broth media for 12 h with subsequent survival recovery on agar plates. Additionally, agar plates were precoated with ILP100 at clinical doses (3 vs. 24 h, 1 x 10(7) CFU/cm(2)) followed by co-culture with pathogens inoculated in soft agar (1 x 10(4 )CFU/cm(2)). To compare ILP100 with relevant antibiotics, MDR-inoculated soft agar was applied to plates with standardized ILP100 drops and antibiotic-loaded discs, followed by 18-20 h aerobic incubation at 37 degrees C. Results: Dose-dependent growth inhibition of all pathogens was demonstrated, as 1000:1 and 100:1 (ILP100/isolate) inhibited pathogenic growth up to log 6.4 and log 4.3 CFU/ml, respectively. Potent antimicrobial effects were demonstrated after precoating with ILP100, as pathogen recovery was only demonstrated after 3 h of precoating, only for 10/18 isolates and then only partially. Benchmarking to relevant antibiotic discs resulted in large cleared zones surrounding the ILP100 spots but not the antibiotic discs, demonstrating potent bacterial killing by ILP100-secreted factors. Interestingly, the MDR pathogens were significantly more sensitive to the ILP100 released factors than the non-MDR isolates. Conclusion: ILP100 effectively eliminates MDR wound pathogens, which reveals a promising strategy for the development of new classes of urgently needed antimicrobials.