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Simultaneous Rapid Detection and Serotyping of Cronobacter sakazakii Serotypes O1, O2, and O3 by Using Specific
Eva J Scharinger1, Richard Dietrich1, Ina Kleinsteuber1
1Department of Veterinary Science, Faculty of Veterinary Medicine, Ludwig-Maximilians-Universität München, Oberschleißheim, Germany.
Insights
New monoclonal antibodies (MAbs) enable rapid, serotype-specific detection of Cronobacter sakazakii in infant formula. This breakthrough aids in identifying the foodborne pathogen, crucial for preventing infant infections.
Area of Science:
- Food Microbiology
- Immunology
- Diagnostic Development
Background:
- Cronobacter sakazakii is a dangerous foodborne pathogen, frequently linked to infant infections via powdered infant formula (PIF).
- Existing detection methods lack serotype specificity, hindering rapid identification of critical strains (O1, O2, O3) often found in clinical and PIF samples.
Purpose of the Study:
- To develop serotype-specific monoclonal antibodies (MAbs) for Cronobacter sakazakii (serotypes O1, O2, O3).
- To establish sensitive and specific enzyme immunoassays (EIAs) for rapid detection and serotyping of C. sakazakii in food matrices.
Main Methods:
- Generation and characterization of monoclonal antibodies (MAbs) targeting C. sakazakii serotypes O1, O2, and O3.
- Utilized indirect EIAs, immunofluorescence, motility assays, and immunoblotting to identify antigenic targets (LPS and EPS).
- Developed and validated sandwich EIAs for detecting C. sakazakii in artificially contaminated PIF samples.
Main Results:
- Successfully created MAbs specific to C. sakazakii serotypes O1, O2, and O3, targeting lipopolysaccharide (LPS) and exopolysaccharide (EPS).
- Developed highly sensitive sandwich EIAs capable of detecting 2 × 10^3 to 9 × 10^6 CFU/ml, with high inclusivity for target serotypes (93% O1, 100% O2, 87% O3).
- Demonstrated high specificity, with minimal cross-reactivity, and successful detection of C. sakazakii in PIF samples with low contamination levels after pre-enrichment.
Conclusions:
- The developed serotype-specific MAbs and sandwich EIAs offer a reliable and rapid method for detecting and simultaneously serotyping Cronobacter sakazakii.
- This advancement is critical for improving food safety, particularly in the analysis of powdered infant formula, and mitigating risks associated with this pathogen.
Abstract:
Cronobacter sakazakii is a foodborne pathogen associated with rare but often lethal infections in neonates. Powdered infant formula (PIF) represents the most frequent source of infection. Out of the identified serotypes (O1 to O7), O1, O2, and O3 are often isolated from clinical and PIF samples. Serotype-specific monoclonal antibodies (MAbs) suitable for application in enzyme immunoassays (EIAs) for the rapid detection of C. sakazakii have not yet been developed. In this study, we created specific MAbs with the ability to bind toC. sakazakii of serotypes O1, O2, and O3. Characterization by indirect EIAs, immunofluorescence, motility assays, and immunoblotting identified lipopolysaccharide (LPS) and exopolysaccharide (EPS) as the antigenic determinants of the MAbs. The established sandwich EIAs were highly sensitive and were able to detect between 2 × 10(3)and 9 × 10(6)CFU/ml. Inclusivity tests confirmed that 93% of serotype O1 strains, 100% of O2 strains, and 87% of O3 strains were detected at low cell counts. No cross-reactivity with >100 strains of Cronobacter spp. and other Enterobacter iaceae was observed, except for that with C. sakazakii serotype O3 and Cronobacter muytjensii serotype O1. Moreover, the sandwich EIAs detected C. sakazakii in PIF samples artificially contaminated with 1 to 10 bacterial cells per 10 g of sample after 15 h of preenrichment. The use of these serotype-specific MAbs not only allows the reliable detection of C. sakazakii strains but also enables simultaneous serotyping in a simple sandwich EIA method.
