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Updated: May 5, 2026
Antimicrobial Characterization of Advanced Materials for Bioengineering Applications
Published on: August 4, 2018
Modified in-vitro AATCC-100 procedure to measure viable bacteria from wound dressings
Sang Hyuk Lee1,2,3, Thomas Glover1,2,3, Nathan Lavey2,3
1Department of Chemical and Biomolecular Engineering, Whiting School of Engineering, Johns Hopkins University, Baltimore, MD, United States of America.
Abstract:
Chronic wounds are reoccurring healthcare problems in the United States and cost up to $50 billion annually. Improper wound care results in complications such as wound debridement, surgical amputation, and increased morbidity/ mortality due to opportunistic infections. To eliminate wound infections, many antimicrobial dressings are developed and submitted to FDA for evaluation. AATCC-100 is a standard method widely used to evaluate cloth wound dressings. This method, requires enrichment, followed by culturing to measure the concentration of culturable organisms; a caveat to this method could result in neglected viable but nonculturable (VBNC) bacteria and overestimate the antimicrobial properties of wound dressings. Therefore, the objectives of this study were to assess this accepted protocol with quantitative real-time polymerase chain reaction (qRT-PCR), to measure time dependent antimicrobial efficacy of wound dressing, and to examine for potential viable bacteria but non-culturable as compared with traditional plating methods. The test organisms included opportunistic pathogens: Pseudomonas aeruginosa (ATCC 15692) and Staphylococcus aureus (ATCC 43300). To mimic a wound dressing environment, samples of commercially available wound dressings (McKesson Inc.) with silver ion (positive control) and dressings without silver ion (positive control) were assessed under sterile conditions. All samples were examined by the original protocol (the extended AATCC-100 method) and qRT-PCR. The expression of specific housekeeping genes was measured (proC for P. aeruginosa and 16s rRNA for S. aureus). Based on these tests, log reduction of experimental conditions was compared to identify time dependent and precise antimicrobial properties from wound dressing samples. These results showed antimicrobial properties of wound dressings diminished as incubation days are increased for both methods from day 1 PCR result of 4.31 ± 0.54 and day 1 plating result of 6.31 ± 3.04 to day 3 PCR result of 1.22 ± 0.97 and day 3 plating result of 5.89 ± 2.41. These results show that data from qRT-PCR generally produced lower standard deviation than that of culture methods, hence shown to be more precise. Complementary parallel analysis of samples using both methods better characterized antimicrobial properties of the tested samples.
Insights
This study reveals quantitative real-time PCR (qRT-PCR) offers a more precise method for evaluating antimicrobial wound dressings than traditional culture methods. It accurately measures the time-dependent efficacy of dressings against bacteria like Pseudomonas aeruginosa and Staphylococcus aureus.
Area of Science:
- Biomedical Engineering
- Microbiology
- Materials Science
Background:
- Chronic wounds pose a significant healthcare burden, costing up to $50 billion annually in the US.
- Improper wound care can lead to severe complications, including opportunistic infections, amputation, and increased mortality.
- Current standard methods for evaluating antimicrobial wound dressings, like AATCC-100, may overestimate efficacy by neglecting viable but non-culturable (VBNC) bacteria.
Purpose of the Study:
- To assess the accuracy of the AATCC-100 protocol using quantitative real-time polymerase chain reaction (qRT-PCR).
- To measure the time-dependent antimicrobial efficacy of wound dressings.
- To compare qRT-PCR with traditional plating methods for detecting viable but non-culturable bacteria.
Main Methods:
- Commercial wound dressings with and without silver ions were tested against Pseudomonas aeruginosa and Staphylococcus aureus.
- Samples were analyzed using both the traditional AATCC-100 method and qRT-PCR, measuring specific bacterial genes (proC and 16s rRNA).
- Log reduction was calculated to compare the time-dependent antimicrobial properties identified by each method.
Main Results:
- Antimicrobial properties of the tested dressings diminished over time, as indicated by both qRT-PCR and plating methods.
- qRT-PCR results showed lower standard deviation compared to culture methods, indicating greater precision.
- Day 1 PCR log reduction was 4.31 ± 0.54, decreasing to 1.22 ± 0.97 by Day 3, while plating showed a reduction from 6.31 ± 3.04 to 5.89 ± 2.41.
Conclusions:
- qRT-PCR provides a more precise and reliable method for assessing the antimicrobial efficacy of wound dressings over time.
- The study highlights the potential for viable but non-culturable bacteria to be missed by traditional methods.
- Parallel analysis using both qRT-PCR and culture methods offers a more comprehensive characterization of wound dressing antimicrobial activity.
