To improve the action of already in use antibiotics or new antimicrobial agents against different bacteria, the development of effective combinations of antimicrobial peptides (AMPs) with enzymes that can quench the quorum (QQ) sensing of bacterial cells was undertaken. Enzymes hydrolyzing N-acyl homoserine lactones (AHLs) and peptides that are signal molecules of Gram-negative and Gram-positive bacterial cells, respectively, were estimated as “partners” for antibiotics and antimicrobial peptides in newly designed antimicrobial–enzymatic combinations. The molecular docking of six antimicrobial agents to the surface of 10 different QQ enzyme molecules was simulated in silico. This made it possible to choose the best variants among the target combinations. Further, bacterial cellulose (BC) was applied as a carrier for uploading such combinations to generally compose prototypes of effective dressing materials with morphology, providing good absorbance. The in vitro analysis of antibacterial activity of prepared BC samples confirmed the significantly enhanced efficiency of the action of AMPs (including polymyxin B and colistin, which are antibiotics of last resort) in combination with AHL-hydrolyzing enzymes (penicillin acylase and His6-tagged organophosphorus hydrolase) against both Gram-negative and Gram-positive cells.

N-acyl homoserine lactones (AHLs) are quorum sensing (QS) signal molecules used by most Gram-negative pathogenic bacteria. In this article the lactonase activity of the preparations based on hexahistidine-tagged organophosphorus hydrolase (His6-OPH) towards AHLs was studied. Initially, three of the most interesting β-lactam antibiotics were selected from seven that were trialed during molecular docking to His6-OPH. Combinations of antibiotics (meropenem, imipenem, ceftriaxone) and His6-OPH taken in the native form or in the form of non-covalent enzyme-polyelectrolyte complexes (EPCs) with poly(glutamic acid) or poly(aspartic acid) were obtained and investigated. The lactonase activity of the preparations was investigated under different physical-chemical conditions in the hydrolysis of AHLs [N-butyryl-D,L-homoserine lactone, N-(3-oxooctanoyl)-D,L-homoserine lactone, N-(3-oxododecanoyl)-L-homoserine lactone]. An increased efficiency of catalytic action and stability of the lactonase activity of His6-OPH was shown for its complexes with antibiotics and was confirmed in trials with bacterial strains. The broadening of the catalytic action of the enzyme against AHLs was revealed in the presence of the meropenem. Results of molecular docking of AHLs to the surface of the His6-OPH dimer in the presence of antibiotics allowed proposing the mechanism of such interference based on a steric repulsion of the carbon chain of hydrolyzed AHLs by the antibiotics bounded to the enzyme surface.

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The effect of Bacitracin as an antibiotic acting against Gram-positive bacterial cells was evaluated in combination with hexahistidine-containing organophosphate hydrolase (His6-OPH), possessing lactonase activity against various N-acylhomoserine lactones produced by most Gram-negative bacteria as quorum-sensing molecules. The molecular docking technique was used to obtain in silico confirmation of possible interactions between molecules of His6-OPH and Bacitracin as well as the absence of a significant influence of such interactions on the enzymatic catalysis. The in vitro experiments showed a sufficient catalytic efficiency of action of the His6-OPH/Bacitracin combination as compared to the native enzyme. The notable improvement (up to 3.3 times) of antibacterial efficiency of Bacitracin was revealed in relation to Gram-negative bacteria when it was used in combination with His6-OPH. For the first time, the action of the Bacitracin with and without His6-OPH was shown to be effective against various yeast strains, and the presence of the enzyme increased the antibiotic effect up to 8.5 times. To estimate the role of the enzyme in the success of His6-OPH/Bacitracin with yeast, in silico experiments (molecular docking) with various fungous lactone-containing molecules were undertaken, and the opportunity of their enzymatic hydrolysis by His6-OPH was revealed in the presence and absence of Bacitracin.

In the present work, innovative composite biomaterials possessing bactericidal properties and based on the hexahistidine-tagged organophosphorus hydrolase (His6-OPH) entrapped in the poly(vinyl alcohol) cryogel (PVA-CG)/bacterial cellulose (BC) were developed. His6-OPH possesses lactonase activity, with a number of N-acyl homoserine lactones being the inducers of Gram-negative bacterial resistance. The enzyme can also be combined with various antimicrobial agents (antibiotics and antimicrobial peptides) to improve the efficiency of their action. In this study, such an effect was shown for composite biomaterials when His6-OPH was entrapped in PVA-CG/BC together with β-lactam antibiotic meropenem or antimicrobial peptides temporin A and indolicidin. The residual catalytic activity of immobilized His6-OPH was 60% or more in all the composite samples. In addition, the presence of BC filler in the PVA-CG composite resulted in a considerable increase in the mechanical strength and heat endurance of the polymeric carrier compared to the BC-free cryogel matrix. Such enzyme-containing composites could be interesting in the biomedical field to help overcome the problem of antibiotic resistance of pathogenic microorganisms.