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Rapid evaluation of the mycobactericidal efficacy of disinfectants in the quantitative carrier test EN 14563 by using
Katrin Steinhauer1, Iris Eschenbacher, Nadine Radischat
1Schülke & Mayr GmbH, Department of Product Development Hygiene/Bioscience, Robert-Koch-Str. 2, 22851 Norderstedt, Germany. katrin.steinhauer@schuelke.com
Abstract:
To prevent transmission of mycobacterial pathogens, medical devices must be disinfected by germicides with proven mycobactericidal activity. The quantitative carrier test EN 14563 provides an international standard for evaluation of the mycobactericidal activity of disinfectants under practical conditions. However, tests according to the EN 14563 standard are based on cultivation, and results are available only after 21 days. The aim of this study was to accelerate assessment of dosage and contact times of mycobactericidal preparations based on the EN 14563 standard. To this end, a gfp gene was constructed with a codon usage adapted for Mycobacterium tuberculosis. Expression of the gfp(m)(2+) gene in Mycobacterium terrae improved the detection sensitivity by 10-fold over that with a previously used reporter strain. Peracetic acid and a cation-active formulation were tested as commercially available disinfectants for medical devices. M. terrae expressing gfp(m)(2+) was used to determine dosage and contact times for the two test germicides. Fluorescence measurements correlated well with growth of the reporter strain, demonstrating that the fluorescence reliably indicated the number of viable cells. The fluorescence enabled us to determine the mycobactericidal efficacy of the test disinfectants according to the quantitative carrier test EN 14563 standard within at least 15 days. In conclusion, this study establishes gfp(m)(2+)-expressing M. terrae as a new reporter strain for reliable evaluation of mycobactericidal activities of disinfectants with a superior sensitivity and in a significantly shorter time than previously possible.
Insights
This study developed a faster method to test disinfectant effectiveness against mycobacteria. Using a genetically modified bacterium, researchers significantly reduced the time needed to confirm mycobactericidal activity, improving medical device safety.
Area of Science:
- Microbiology
- Disinfection Science
- Medical Device Sterilization
Background:
- Mycobacterial pathogens pose a risk of transmission via contaminated medical devices.
- Standardized quantitative carrier tests (EN 14563) evaluate disinfectant mycobactericidal activity but require 21 days for results.
- Accelerating the assessment of disinfectant efficacy is crucial for timely medical device reprocessing.
Purpose of the Study:
- To develop a method for accelerating the assessment of disinfectant dosage and contact times for mycobactericidal preparations.
- To adapt the EN 14563 standard for faster evaluation of disinfectant efficacy.
- To establish a sensitive reporter strain for rapid detection of mycobactericidal activity.
Main Methods:
- Construction of a green fluorescent protein (GFP) gene with codon usage optimized for Mycobacterium tuberculosis.
- Expression of the modified GFP gene (gfp(m)(2+)) in Mycobacterium terrae to create a sensitive reporter strain.
- Testing of peracetic acid and a cation-active formulation using the gfp(m)(2+)-expressing M. terrae strain and fluorescence measurements.
Main Results:
- The gfp(m)(2+) reporter strain demonstrated a 10-fold improvement in detection sensitivity compared to previous strains.
- Fluorescence measurements correlated strongly with bacterial growth, reliably indicating viable cell counts.
- Mycobactericidal efficacy testing using the new method yielded results within 15 days, significantly faster than the standard 21 days.
Conclusions:
- The gfp(m)(2+)-expressing M. terrae strain provides a sensitive and rapid method for evaluating disinfectant mycobactericidal activity.
- This approach significantly reduces the time required for disinfectant efficacy testing according to the EN 14563 standard.
- The developed reporter strain facilitates quicker assessment of disinfectants, enhancing medical device safety and reprocessing efficiency.
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