Search for effective bacteriolytic enzymes and their combinations for the treatment of polymicrobial biofilm-associated infections.

Polymicrobial biofilms resistant to antibiotic therapy are often associated with different severe human diseases, including skin and soft tissue injuries, local mucosal and dental infections, post-traumatic and chronic osteomyelitis, and implant-associated infections. A possible treatment option for these challenging infections may be the use of enzybiotics containing bacteriolytic enzymes (or lysins). However, elimination of polymicrobial infections requires an effective combination of enzymes to expand the narrow activity spectrum of individual lysins and to ensure successful treatment. We screened ten bacteriolytic enzymes for their antibacterial and biochemical properties, including effective doses, rate of action, spectrum of action, as well as effects of pH, temperature, and buffer systems on enzymes activity. The selected Gram-negative bacteria-targeting enzyme was further modified with an antimicrobial peptide to improve its stability. This engineered lysin GRC-ML07 was studied in combination with Gram-positive bacteria-targeting enzymes. Our results convincingly demonstrate that combinations of lysins with different substrate specificity and Gram specificity result in synergistic and additive effects against pathogenic bacteria, such as Staphylococcus aureus, Pseudomonas aeruginosa, and Enterobacterales. Importantly, this was shown for both planktonic cells and biofilms, including polymicrobial consortia, as combinations significantly decreased bactericidal concentrations of enzymes against Gram-positive and Gram-negative bacteria and improved antibiofilm properties. This novel antibacterial enzybiotic composition was also safe in in vitro experiments.