Method for detection of Enterobacter sakazakii in milk - Master's thesis - Dissertation

Enterobacter sakazakii, also known as *Enterobacter sakazakii*, is a Gram-negative bacillus that lacks flagella, motility, and spores. It was previously classified under *Enterobacter cloacae* but was reclassified in 1980. This bacterium is particularly dangerous for neonates, as it can cause severe infections such as meningitis, enterocolitis, and sepsis, with a mortality rate ranging from 40% to 80%. Since its first reported case in 1961, outbreaks have been documented globally in countries like Denmark, Greece, the UK, the Netherlands, and Iceland. Due to its high risk, the World Health Organization has recognized it as a critical pathogen linked to infant deaths. In China, regulations were introduced in 2005 to detect *E. sakazakii* in infant formula, ensuring that no harmful bacteria are present in these products. First, let's explore the biological characteristics of *E. sakazakii*. Morphologically, it is a Gram-negative bacillus with pili, no spores, and no motility. It grows on various media such as nutrient agar, blood agar, MacConkey agar, EMB agar, and deoxycholic acid agar. Its optimal growth temperature ranges from 25°C to 36°C, though some strains can grow at temperatures up to 47°C. After 24 hours of incubation at 25–36°C, yellow colonies appear on TSA and BHIA plates, while purple-red colonies are seen on VRBG agar. These distinct features help in initial identification. Next, traditional detection methods involve several steps. A sample is first enriched in buffered peptone water and then incubated in mLST-Vm broth. After incubation, suspicious colonies are transferred to chromogenic agar plates. Biochemical tests, such as using commercial kits or automated systems, are then used to confirm the presence of *E. sakazakii*. The results are reported based on colony morphology and biochemical profiles, with the detection limit set at 100g/mL of sample. To improve efficiency, new technologies have emerged. One method uses a fluorescent selective medium containing α-MUG, which allows *E. sakazakii* to produce yellow colonies that fluoresce under UV light. Another approach involves measuring absorbance at 405 nm after hydrolyzing p-nitrophenol-α-D-glucoside, indicating the presence of α-glucosidase activity. Additionally, fluorescence quantitative PCR has revolutionized detection by enabling rapid and accurate identification within 2 days, compared to the traditional 6–7 days. This method has shown excellent specificity, detecting 68 strains of *E. sakazakii* without false positives or negatives. In conclusion, while traditional methods remain reliable, they are time-consuming and labor-intensive. Newer techniques offer faster, more accurate, and efficient alternatives. As research continues, these advancements will play a crucial role in improving food safety and protecting vulnerable populations like infants.

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