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Updated: Jun 10, 2025
Biosensor for Detection of Antibiotic Resistant Staphylococcus Bacteria
Published on: May 8, 2013
Phage LysSA163-CBD mediated specific recognition coupled with ATP bioluminescence for the sensitive detection of
Peng Guan1, Ruining Li1, Yifeng Ding1
1Key Laboratory of Environment Correlative Dietology, Huazhong Agricultural University, Wuhan, 430070, China; College of Food Science and Technology, Huazhong Agricultural University, Wuhan, 430070, China.
Background:
Staphylococcus aureus is a significant foodborne pathogen, commonly detected in milk and meat products. Conventional detection methods have limited sensitivity and specificity, which are time-consuming and susceptible to background interference from complex samples, and cannot effectively distinguish live and dead bacteria.
Results:
Herein, we developed a novel adenosine triphosphate (ATP) bioluminescence method coupled with magnetic separation, which is based on phage-encoded endolysin LysSA163-CBD (CBD 163) for rapid and specific detection of viable Staphylococcus aureus. The expressed protein (CBD 163) was derived from the phage LSA2301 and was successfully expressed in Escherichia coli BL21 following an induction period of 4 h at 37 °C, with a molecular weight approximating 40 kDa. The optimal conditions for CBD-magnetic beads (cMBs) to capture S. aureus cells were determined to be 100 μL/mL cMBs at 25 °C for 30 min. The viable S. aureus cells were disrupted by the Cetyl trimethyl ammonium bromide (CTAB) to release intracellular ATP. Then, the ATP reacted with the firefly luciferase and D-Luciferin-based bioluminescence (BL) reagents solution to generate intensive BL signal. The CBD-magnetic separation-ATP bioluminescence (cMS-BL) assay was able to quickly detect viable S. aureus via ATP bioluminescence in 45 min, with a detection range from 5 × 103 to 5 × 107 CFU/mL and limit of detection (LOD) of 190 CFU/mL. Additionally, the cMS-BL method exhibited high specificity and anti-interference ability, which has been successfully applied to quantify S. aureus cells in crayfish-tail, chicken, and skim milk.
Significance And Novelty:
These results demonstrate the potential of CBD 163 as a versatile and robust biorecognition element for rapid and specific detection of viable S. aureus in food matrices. The proposed phage-derived bacteria-binding proteins-based protocol for BL detection shows various advantages, including high sensitivity, simple operation, and the capability to distinguish live bacteria, providing a strategy for designing high-quality biorecognition element toward foodborne pathogens.
Insights
A new phage-derived endolysin method rapidly detects viable Staphylococcus aureus in food. This magnetic separation-ATP bioluminescence assay offers high sensitivity and specificity for pathogen detection.
Area of Science:
- Microbiology
- Biotechnology
- Food Science
Background:
- Staphylococcus aureus is a major foodborne pathogen found in milk and meat.
- Conventional detection methods lack sensitivity, specificity, and cannot differentiate live/dead bacteria.
- Existing methods are time-consuming and prone to interference in complex food samples.
Purpose of the Study:
- To develop a rapid, specific method for detecting viable Staphylococcus aureus.
- To utilize phage-derived endolysin LysSA163-CBD for bacterial cell capture.
- To couple magnetic separation with adenosine triphosphate (ATP) bioluminescence for enhanced detection.
Main Methods:
- Expressed and purified phage-encoded endolysin LysSA163-CBD (CBD 163).
- Developed CBD-magnetic beads (cMBs) for specific capture of S. aureus.
- Employed Cetyl trimethyl ammonium bromide (CTAB) to release intracellular ATP for bioluminescence detection.
Main Results:
- The CBD-magnetic separation-ATP bioluminescence (cMS-BL) assay detected viable S. aureus within 45 minutes.
- Achieved a detection range of 5 × 10³ to 5 × 10⁷ CFU/mL with a limit of detection (LOD) of 190 CFU/mL.
- Successfully applied the cMS-BL method to quantify S. aureus in crayfish-tail, chicken, and skim milk samples.
Conclusions:
- CBD 163 is a versatile biorecognition element for rapid, specific detection of viable S. aureus.
- The phage-derived protein-based bioluminescence assay offers high sensitivity and distinguishes live bacteria.
- This method provides a robust strategy for developing improved detection tools for foodborne pathogens.
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