Ericksen, B. | Search bioRxiv

bioRxiv2026

Virtual colony count study of the inoculum effect of HNP1 against Staphylococcus aureus ATCC 29213

BackgroundVirtual colony count is a kinetic, 96-well turbidimetric assay that has been used since 2003 to determine the antimicrobial activity of antimicrobial peptides including the defensin HNP1. Virtual colony count results differed from traditional colony counting results in studies of the antimicrobial activity of the human cathelicidin LL-37 and related peptides. The difference could possibly have been caused by an inoculum effect. MethodsThe virtual colony count assay was conducted using inocula that varied from 1250 to 1x108 virtual colony forming units (CFUv) per milliliter. ResultsThe virtual colony count assay demonstrated a pronounced inoculum effect of HNP1 against Staphylococcus aureus ATCC 29213, accompanied by biofilm formation observed in the wells of the 96 well plates at all inocula. The S. aureus inoculum effect was not as drastic as previously reported for Escherichia coli. ConclusionsThe inoculum effect is further evidence that biofilm formation is a resistance mechanism used by a variety of bacteria against antimicrobial peptides such as HNP1.

microbiology↗

bioRxiv2022

Enhancement of the Activity of the Antimicrobial Peptides HNP1 and LL-37 by Bovine Pancreatic Ribonuclease A

BackgroundHNP1, LL-37, and HBD1 are antimicrobial against Escherichia coli ATCC 25922 at the standard inoculum but less active at higher inocula. MethodsThe virtual colony count (VCC) microbiological assay was adapted for high inocula and the addition of yeast tRNA and bovine pancreatic ribonuclease A (RNase). 96-well plates were read for 12 hours in a Tecan Infinite M1000 plate reader and photographed under 10x magnification. ResultsAdding tRNA 1:1 to HNP1 at the standard inoculum almost completely abrogated activity. Adding RNase 1:1 to HNP1 at the standard inoculum of 5x105 CFU/mL did not enhance activity. Increasing the inoculum to 6.25x107 CFU/mL almost abrogated HNP1 activity. However, adding RNase 25:1 to HNP1 enhanced activity. Adding both tRNA and RNase resulted in enhanced activity, indicating that the enhancement effect of RNase overwhelms the inhibiting effect of tRNA when both are present. HBD1 activity at the standard inoculum was almost completely abrogated by the addition of tRNA, but LL-37 activity was only slightly inhibited by tRNA. At the high inoculum, LL-37 activity was enhanced by RNase. HBD1 activity was not enhanced by RNase. RNase was not antimicrobial in the absence of antimicrobial peptides. Cell clumps were observed at the high inoculum in the presence of all three antimicrobial peptides and at the standard inoculum in the presence of HNP1+tRNA. ConclusionsAntimicrobial peptide-ribonuclease combinations have the potential to be active against high cell concentrations and biofilms, conditions where the antimicrobial agent alone is relatively ineffective.

microbiology↗