Abstract
The extremely rapid spread of multiple-antibiotic resistance among Gram-negative pathogens threatens to move humankind into the so-called “post-antibiotic era” in which the most efficient and safe antibiotics will not work. Bacteriophage lysins represent promising alternatives to antibiotics, as they are capable of digesting bacterial cell wall peptidoglycans to promote their osmotic lysis. However, relatively little is known regarding the spectrum of lysin bactericidal activity against Gram-negative bacteria. In this study, we present the results of in vitro activity assays of three putative and newly cloned Myoviridae bacteriophage endolysins (LysAm24, LysECD7, and LysSi3). The chosen proteins represent lysins with diverse domain organization (single-domain vs. two-domain) and different predicted mechanisms of action (lysozyme vs. peptidase). The enzymes were purified, and their properties were characterized. The enzymes were tested against a panel of Gram-negative clinical bacterial isolates comprising all Gram-negative representatives of the ESKAPE group. Despite exhibiting different structural organizations, all of the assayed lysins were shown to be capable of lysing Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniae, Escherichia coli, and Salmonella typhi strains. Less than 50 μg/mL was enough to eradicate growing cells over more than five orders of magnitude. Thus, LysAm24, LysECD7, and LysSi3 represent promising therapeutic agents for drug development.
Highlights
Antibiotic microbial resistance (AMR) is a natural aspect of microbe evolution under selective pressure
The bacterial strains used in the study included both reference strains and clinical isolates of Gram-negative representatives of the ESKAPE group of pathogens, including A. baumannii, P. aeruginosa, K. pneumoniae, E. coli, and S. typhi as well as Staphylococcus haemolyticus and S. aureus from the collection of the N.F
BL21(DE3) pLysS expression strain, extensive cells lysis of these cells was not detected during the expression and purification procedures, indicating that either internal cell membrane penetration did not take place or that lysis of the expression strain occurred to some degree with no significant effect on the efficiency of protein production
Summary
Antibiotic microbial resistance (AMR) is a natural aspect of microbe evolution under selective pressure. Because most antibiotics currently in use have natural analogues with similar native structures, AMR-associated genes of environmental bacteria can be distributed to clinically important strains through horizontal gene transfer [1,2]. Viruses 2019, 11, 284 pathogens of the ESKAPE group (Enterococcus faecium, Staphylococcus aureus, Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae, and other Enterobacteriaceae species) These pathogens represent the greatest threat among the so-called superbugs, which can rapidly acquire resistance to several classes of antibiotics and are able to cause a variety of nosocomial infections, such as bacteremia, pneumonia, and wound and skin infections [3,8]. Virus-based drugs have significant drawbacks, including their labor-intensive acquisition and associated stringent safety requirements; they are unstable during storage and need constant pharmacokinetic monitoring [11,12]
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