Sensitive and Selective Detection of Enterococcus faecalis Using a New Turn-on Fluorogenic β-glucosidase Substrate...

Area of Science:

• Biochemistry
• Analytical Chemistry
• Microbiology

Background:

• Developing sensitive and selective probes for enzyme activity and bacterial detection is crucial in diagnostics.
• Existing methods may lack the sensitivity or specificity required for early-stage detection of specific bacteria like Enterococcus faecalis.
• 7-hydroxycoumarin derivatives offer potential as fluorescent probes due to their optical properties.

Purpose of the Study:

• To develop a novel, simple-to-synthesize, turn-on fluorogenic substrate (CFMU-Glu) for β-glucosidase activity.
• To create a sensitive and selective fluorogenic broth (PBF-B) for the detection of Enterococcus faecalis (E. faecalis).
• To establish a rapid and effective method for detecting E. faecalis in various sample types.

Main Methods:

• Synthesis of a new fluorogenic substrate, CFMU-Glu, based on a 7-hydroxycoumarin derivative (CFMU).
• Optimization of detection conditions, including pH, for almond β-glucosidase and E. faecalis using CFMU-Glu.
• Development of a fluorogenic broth (PBF-B) incorporating CFMU-Glu and polymyxin B for enhanced E. faecalis detection.
• Evaluation of detection limits using fluorescence microplate reader and naked eye observation.
• Testing the method's efficacy in real-world samples like drinking water, herbal tea, and milk.

Main Results:

• CFMU-Glu was successfully synthesized and demonstrated sensitive turn-on fluorescence for β-glucosidase activity.
• Optimal pH for almond β-glucosidase and E. faecalis detection with CFMU-Glu were determined as 6.50 and 7.00, respectively.
• The PBF-B broth significantly improved detection selectivity and signal intensity for E. faecalis.
• Low detection limits were achieved: 5.01 × 10⁴ CFU mL⁻¹ (fluorescence reader) and 1.0 × 10⁵ CFU mL⁻¹ (naked eye) for fluorogenic detection.
• A chromogenic detection limit of 1.0 × 10⁶ CFU mL⁻¹ (naked eye) was also established.
• The method successfully detected as low as 8 CFU mL⁻¹ of E. faecalis in drinking water, herbal tea, and milk within 10 hours, without pre-enrichment.

Conclusions:

• CFMU-Glu is a versatile and sensitive fluorogenic substrate for β-glucosidase activity and E. faecalis detection.
• The PBF-B broth, combined with CFMU-Glu and polymyxin B, offers a highly sensitive and selective method for E. faecalis detection.
• The developed method provides rapid, pre-enrichment-free detection of E. faecalis in diverse samples, with potential applications in food safety and clinical diagnostics.