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 to C. 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 Enterobacteriaceae 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. Cronobacter spp. are Gram-negative opportunistic foodborne pathogens of the family Enterobacteriaceae that cause rare but severe infections in patients of all age groups. In adults, Cronobacter spp. are often associated with nosocomial infections, including pneumonia, septicemia, wound infections, and osteomyelitis, while causing invasive disease in young infants and neonates (1-4). Among the seven identified Cronobacter species, C. sakazakii, C. malonaticus, C. muytjensii, C. turicensis, C. dublinensis, C. condimenti, and C. universalis (5-8), C. sakazakii plays a prominent role due to it causing life-threatening infections in neonates (9-11). Clinically manifested infections present as necrotizing enterocolitis, sepsis, and meningitis, with a mortality rate as high as 80% (1, 12, 13). Although C. sakazakii has been isolated from a variety of different plant-and animal-based food products (14, 15), the presence in powdered infant formula (PIF) seems crucial in the infection of neonates (9, 12, 16). According to an established O-antigen serotyping scheme based on rabbit antisera and a PCRbased serotyping method (17-21), seven serotypes (O1 to O7) have been identified for C. sakazakii. Serotypes O1 and O2 seem to be most prevalent in PIF samples and in clinical cases, whereas serotype O3 has been isolated quite frequently from PIF but not as often from clinical cases (19,(22)(23)(24).Today, the contamination of PIF by C. sakazakii is being detected using conventional microbiological m...

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

“… a Using specific MAbs 1C4 (O1), 2F8 (O2) (Scharinger et al, 2016 ) . b Reactivity of overnight culture (LB medium) .…”

“…MAbs 1C4 and 2F8 specific to C. sakazakii serotypes O1 and O2 respectively were described in a previous study (Scharinger et al, 2016 ).…”

“…At present, the species C. sakazakii consists of five serotypes O1–O4 and O7 (Blažková et al, 2015 ). Predominantly the serotypes O1 and O2 are isolated from clinical as well as from food products, suggesting that some serotypes of C. sakazakii might be more virulent than others (Blažková et al, 2015 ; Yan et al, 2015a , b ; Scharinger et al, 2016 ). Therefore, it is important for epidemiological studies to not only identify the genus and species but also the serotype of the pathogen.…”

“…However, these methods are laborious and definitive identification of Cronobacter species–without serotyping–takes up to 6 days, because diverse enrichments and biochemical confirmation steps are required. Besides culture-based methods, other rapid, highly sensitive and specific approaches for Cronobacter detection such as fluorescence in situ hybridization (FISH) (Almeida et al, 2009 ), DNA microarray (Wang et al, 2009 ), polymerase chain reaction (PCR) (Zimmermann et al, 2014 ; Yan and Fanning, 2015 ; Zhou et al, 2016 ), and antibody-based immunoassays (Hochel and Škvor, 2009 ; Blažková et al, 2011 ; Park et al, 2012 ; Xu et al, 2014 ; Scharinger et al, 2016 ) have been reported. All these techniques require specialized equipment and trained staff, consequently, they become expensive and laboratory-based.…”

“…Among the diagnostic tests, the immunochromatographic strip assays satisfy all these prerequisites and some lateral flow tests for different pathogenic bacteria or their agents have already been successfully developed in order to enhance food safety (Kawatsu et al, 2008 ; Fang et al, 2014 ; Cox et al, 2015 ; Wu et al, 2015 ; Raeisossadati et al, 2016 ). Therefore, the aim of this study was to develop a multiplexed lateral flow assay (LFA) for rapid detection and simultanous serotyping of C. sakazakii serotypes O1 and O2, based on the recently described highly serotype specific monoclonal antibodies (MAbs) (Scharinger et al, 2016 ). The ability of the established lateral flow test strip to specifically detect C. sakazakii was confirmed by analyzing artificially contaminated PIF samples after enrichment in buffered peptone water (BPW) in comparison to the standard ISO reference method.…”