Detection of Cronobacter Genus in Powdered Infant Formula by Enzyme-linked Immunosorbent Assay Using Anti-Cronobacter Antibody

Xinjie Song,Myunghee Kim,Shruti Shukla,Sunhyun Park,Gibaek Lee

doi:10.3389/fmicb.2016.01124

Abstract

Cronobacter species (Cronobacter spp.) are hazardous foodborne pathogens associated with baby food, powdered infant formula (PIF). To develop a rapid and sensitive method for simultaneous detection of seven Cronobacter spp. in PIF, an indirect non-competitive enzyme-linked immunosorbent assay (INC-ELISA) was developed based on a novel immunoglobulin G (IgG), anti-Cronobacter IgG. The developed INC-ELISA was able to detect seven Cronobacter spp. at concentrations ranging from (5.6 ± 0.30) × 103 to (2.1 ± 0.01) × 105 colony forming unit (CFU)/mL in pure culture. Further, INC-ELISA employing anti-Cronobacter IgG was applicable for analysis of PIF samples contaminated with less than <10 cells of Cronobacter spp. per 25 g of PIF in 36 h. The developed antibody showed slight cross-reactivity with Franconibacter pulveris (LMG 24057) at high concentration (108 CFU/mL). The INC-ELISA method displayed excellent specificity without compromising cross-reactivity with other foodborne pathogens. The INC-ELISA assay method developed in this study using a novel anti-Cronobacter IgG facilitated highly sensitive, efficient, and rapid detection of Cronobacter spp. in baby food.

Highlights

Bacteria of the genus Enterobacter are increasingly important human pathogens that cause neonatal meningitis and sepsis (Hunter and Bean, 2013)
Other strains used in this study were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA) and Korean Culture Center of Microorganisms (KCCM; Seoul, Korea)
For antibodies developed using Formalin-killed cell (FKC) and sonicated cell protein (SCP) mixtures of Cronobacter spp., titers of the developed rabbit anti-Cronobacter serum containing immunoglobulin G (IgG) were determined by INC-enzyme-linked immunosorbent assay (ELISA) after 18 weeks of immunization, as shown in Figures 2 and 3

Summary

Introduction

Bacteria of the genus Enterobacter are increasingly important human pathogens that cause neonatal meningitis and sepsis (Hunter and Bean, 2013). Enterobacter sakazakii has recently been reclassified as a Cronobacter (Strydom et al, 2012). Cronobacter species (Cronobacter spp.), previously known as E. sakazakii, are foodborne pathogens that pose a high risk of infection to neonates as well as immuno-compromised individuals (World Health Organization [WHO], 2007). Cronobacter spp. affect the central nervous system of infants, and survivors often suffer from severe neurological impairments such as hydrocephalus, quadriplegia, and developmental delays (Lai, 2001; Strydom et al, 2012; Jaradat et al, 2014). Species level classification of the Cronobacter genus, which includes C. sakazakii, C. muytjensii, C. malonaticus, C. turicensis, C. dublinensis, C. universalis, and C. condimenti, was recently defined by Iversen et al (2008)

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Results

Conclusion