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

2026 · doi:10.64898/2026.04.09.717392
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Abstract

Background Virtual 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. Methods The virtual colony count assay was conducted using inocula that varied from 1250 to 1×10 8 virtual colony forming units (CFUv) per milliliter. Results The 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 . Conclusions The inoculum effect is further evidence that biofilm formation is a resistance mechanism used by a variety of bacteria against antimicrobial peptides such as HNP1.
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Abstract

Background Virtual 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.

Methods

The virtual colony count assay was conducted using inocula that varied from 1250 to 1×108 virtual colony forming units (CFUv) per milliliter.

Results

The 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.

Conclusions

The inoculum effect is further evidence that biofilm formation is a resistance mechanism used by a variety of bacteria against antimicrobial peptides such as HNP1. Competing Interest Statement The authors have declared no competing interest.

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License: CC-BY-4.0