Comparative evaluation of antimicrobial activity of human granulysin, bovine and porcine NK-lysins against Shiga toxin-producing Escherichia coli O157:H7

Authors: BIERNBAUM, ERIKA - Oak Ridge Institute For Science And Education (ORISE); Dassanayake, Rohana; Nicholson, Eric; Kudva, Indira

Submitted to: PLOS ONE
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/16/2023
Publication Date: 9/28/2023
Citation: Biernbaum, E.N., Dassanayake, R.P., Nicholson, E.M., Kudva, I.T. 2023. Comparative evaluation of antimicrobial activity of human granulysin, bovine and porcine NK-lysins against Shiga toxin-producing Escherichia coli O157:H7. PLOS ONE. 18(9): e0292234. https://doi.org/10.1371/journal.pone.0292234.
DOI: https://doi.org/10.1371/journal.pone.0292234

Interpretive Summary: Shiga toxin-producing Escherichia coli O157:H7 (O157), a foodborne pathogen, causes disease ranging from watery to bloody diarrhea which can progress to serious systemic disease. Cattle harbor O157 in their intestines and cattle feces is a primary source of human infection via ingestion of fecal-contaminated food products. Strategies to reduce O157 survival, particularly in cattle are needed. Hence, we evaluated naturally occurring compounds with antimicrobial properties for their ability to kill O157 in culture. The compounds, referred to as antimicrobial peptides, were able to kill O157 without having a major impact on animal blood cells or causing O157 to produce harmful toxins. Overall, our results identified a potential for the peptides, which were derived from bovine and porcine immune cells, to be further developed into novel non-antibiotic agents to reduce O157 in cattle.

Technical Abstract: Shiga toxin-producing Escherichia coli (STEC) O157:H7 (O157), a foodborne pathogen, causes disease ranging from hemorrhagic colitis to hemolytic uremic syndrome and kidney failure in humans, while remaining harmless to its primary reservoir, cattle. The severity of the human disease associated mainly with Shiga toxin production, and a global emergence of antibiotic resistant STEC highlights the need for effective, non-antibiotic, pre-harvest strategies to reduce O157 in cattle, the principal source of human infection. Towards this goal, three synthetic antimicrobial peptides (AMPs), human granulysin (hGRNL), bovine NK-lysin (bNK2A), and porcine NK-lysin (pNKL), were tested in vitro against O157 isolates. As expected, circular dichroism spectroscopy findings were consistent with a predominantly a-helical conformation for all three AMPs in an environment mimicking bacterial outer surface or liposaccharides. The minimum inhibitory concentrations (MIC) of hGRNL, bNK2A, and pNKL in Müeller-Hinton broth (Cation-Adjusted), as determined by Clinical and Laboratory Standard Institute broth microdilution method, were 200, 12.5-25, and 6.25 µM, respectively. Minimum bactericidal concentrations were identical to MIC, and the bNK2A and pNKL AMPs did not induce Shiga toxin expression in O157 when tested at MIC. Propidium iodide uptake assay revealed faster O157 membrane damage or killing kinetics with bNK2A and pNKL as compared to hGRNL. Nonetheless, transmission electron microscopy demonstrated that all three AMPs mediated damage to O157 membranes. In contrast, the three AMPs showed minimal cytotoxicity (<2%) against cattle red blood cells at tested concentrations (0.39-50 µM). Overall, our results demonstrate the potential for bNK2A and pNKL to be further developed into novel non-antibiotic agents to reduce O157 shedding in cattle.