A Method to Directly Identify Cronobacter sakazakii in Liquid Medium by MALDI-TOF MS

Area of Science:

• Microbiology
• Analytical Chemistry
• Food Safety

Background:

• Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a key technology for microbial identification.
• Cronobacter sakazakii (C. sakazakii) poses a significant threat in powdered infant formula (PIF) environments due to its high infant mortality rate.
• Traditional MALDI-TOF MS methods for C. sakazakii are often limited to qualitative detection.

Purpose of the Study:

• To develop and optimize a novel, cost-effective liquid spotting pretreatment method for MALDI-TOF MS.
• To enhance the accuracy, reproducibility, and quantitative capabilities for C. sakazakii detection.
• To determine the optimal parameters for the liquid spotting method using response surface methodology.

Main Methods:

• Development of a low-cost liquid spotting pretreatment protocol.
• Optimization of pretreatment parameters (formic acid volume, ultrasound power and time, acetonitrile volume) using response surface methodology.
• Validation of the method's applicability, accuracy, and quantitative potential across different sample types.

Main Results:

• Optimal parameters identified: 25 μL of 70% formic acid, 350 W ultrasound for 3 min, and 75 μL acetonitrile.
• Achieved the highest identification score for C. sakazakii (1926.42 ± 48.497).
• Demonstrated 100% identification accuracy for 70 C. sakazakii strains.
• Established detection limits of 4.1 × 10^1 cfu/mL in environmental samples and 2.72 × 10^3 cfu/mL in PIF samples.

Conclusions:

• The developed liquid spotting method offers a robust, accurate, and reproducible approach for C. sakazakii identification using MALDI-TOF MS.
• This method significantly improves upon traditional qualitative detection, offering quantitative potential.
• The optimized protocol is suitable for detecting C. sakazakii in critical food processing environments like PIF production.