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

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.

FIG 1

Microscopic analysis of live C. sakazakii cells by phase contrast (1), immunofluorescence (2), and overlay mode (3) using serotype-specific MAbs: MHI 21172 (O1) stained with MAb 1C4 (A), MHI 21032 (O2) stained with MAb 2F8 (B), and MHI 21166 (O3) stained with MAb 1A11 (C).

FIG 2

Reactivities of MAbs 1C4, 2F8, and 1A11 with (lipo)polysaccharide preparations of different serotypes of C. sakazakii strains. Lane 1, MHI 21011 (O1); lane 2, MHI 21029 (O2); lane 3, MHI 990 (O3).

FIG 3

Swimming ability of C. sakazakii. Bacterial motility was assayed in soft agar without and with specific MAbs (17 μg/ml). MAb 1C4- and MAb 2F8-containing supernatants of hybridoma cells were diluted in soft agar containing 50% Dulbecco's modified Eagle's medium (DMEM). Purified MAb 1A11 was added in soft agar without DMEM. Motility zones were measured after 8 h of incubation at 37°C. (A) MHI 21001 (O1). (B) MHI 21035 (O2). (C) MHI 990 (O3).

FIG 4

Sensitivity of the sandwich EIA for the detection of C. sakazakii strains MHI 21001 (O1), MHI 977 (O2), and MHI 990 (O3). MAbs 1C4, 2F8, and 1A11, respectively (10 μg/ml), served as solid phase. Bacteria were enriched overnight in LB broth at 37°C and subsequently detected in serial dilutions with 1C4-HRP, 2F8-HRP (1:1,000), and 1A11-HRP (1:2,000), respectively.

FIG 5

Evaluation of the established O2-specific sandwich EIA for the analysis of C. sakazakii in PIF. Shown is a comparison of the detection limit of strain MHI 977 (O2) in pure bacterial preparation in PBS (green line) and in enriched 10% PIF broth (blue line). Inset, absorbance values of buffered peptone water (black dashed line) and 10% PIF-buffered peptone water (black solid line) without Cronobacter (background). All samples were analyzed in 2-fold serial dilutions in PBS–0.1% Tween20 and probed with MAb 2F8 (10 μg/ml) and 2F8-HRP (1:1,000 in 1% caseinate–PBS).

FIG 6

Detection of C. sakazakii serotypes O1, O2, and O3 in PIF samples. For each serotype, 3 strains were used to artificially contaminate 10 g of PIF with bacterial cell counts ranging from 1 to 10 CFU. After 15 h of enrichment, serially diluted samples were directly analyzed in the sandwich EIAs. The experiments were conducted in triplicate, as indicated by the columns. The absorbance values were dependent on the amount of C. sakazakii used for the contamination. The absorbance values for the 10-fold diluted samples ranged from 0.156 to 3.5. The cutoff value (absorbance, ≤0.1) of the EIAs is indicated by a dashed red line.