Multiplexed Lateral Flow Test for Detection and Differentiation of Cronobacter sakazakii Serotypes O1 and O2.

The ubiquitous and opportunistic pathogen Cronobacter sakazakii is responsible for severe meningitis, sepsis, and necrotizing enterocolitis in neonates and infants associated with ingestion of contaminated powdered infant formula (PIF). The current ISO method for isolation and detection of Cronobacter spp. is laborious, time-consuming and expensive. In this study, a multiplexed lateral flow test strip was developed to rapidly detect and simultaneously serotype O1 and O2 C. sakazakii serotypes. The assay is based on two monoclonal antibodies (MAb) that specifically bind to the lipopolysaccharides (LPS) of these pathogens. The test strip provides results very quickly; C. sakazakii could be detected in pure culture within 15 min with a sensitivity of 107 CFU/ml. After non-selective enrichment for 18 h as low as one Cronobacter cell per g PIF could be detected. Moreover, the established lateral flow assay (LFA) offers excellent specificity showing no cross-reactivity with other C. sakazakii serotypes, Cronobacter species or Enterobacteriaceae tested. These characteristics, together with several advantages such as speed, simplicity in performance, low analysis cost, and no requirement of specialized skills or sophisticated equipment make the developed multiplexed LFA suitable for reliable detection and serotyping of C. sakazakii serotypes O1 and O2.

Simultaneous Rapid Detection and Serotyping of Cronobacter sakazakii Serotypes O1, O2, and O3 by Using Specific Monoclonal Antibodies.

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.