Genotypic and phenotypic diversity of Cronobacter species from food and environmental sources

The genus Cronobacter includes food-borne pathogens causing neonatal infections such as meningitis, and necrotizing enterocolitis as well as bacteraemia in immunocompromised adults. Understanding the molecular characterisation, clonality and phenotypic diversity of Cronobacter species is essential to reduce the source of microbial contamination of powdered infant formula (PIF) and other food. Therefore, an improved understanding of the diversity of the genus is warranted. \n \nIn the first part of the study, the 7-loci Multilocus sequence typing (MLST) scheme was applied to investigate the diversity of Cronobacter spp. isolated from food and environmental sources. Twenty-six strains that had not previously been profiled were divided into 21 sequence type (STs), and 9 new STs were identified, which had not been previously reported. Cronobacter strains isolated from food and environmental sources were highly diverse with respect to their ST, particularly those from different sources of food. \n \nThis study is the first to describe the development and application of variable number tandem repeat analysis (VNTRA) typing method for C. sakazakii ST4 strains. Nineteen C. sakazakii ST4 strains, which were widely distributed geographically, temporally and origin of source were profiled. These strains were divided into 15 distinct groups based on the number of tandem repeats of 6 VNTR loci. It was concluded that VNTRA profiling could contribute to further understanding of C. sakazakii ST4 diversity and tracking of infection sources. Of particular interest in this research was the finding that the analysis of the lipopolysaccharide (LPS) profiling using BioNumerics software, (version 7.1) showed great ability to discriminate between strains within the same serotype. Furthermore, the present study developed a multiplex PCR assay targeting capsular polysaccharide genes such as kpsS (K1 and K2) and galE (CA1 and CA2) for the specific detection and rapid identification of K-capsule type and colanic acid type respectively. \n \nAnother important finding was that a strong correlation between the amount of mucoid production, type and ratio of monosaccharides production and type of O-antigen serotype. This study indicated also that rhamnose is the main sugar in C. sakazakii serotype O:2 strain. The most interesting observation was that C. sakazakii strains with serotype O:1 and O:4 had high numbers of sublethally injured cells after desiccation, while strains with serotype O:2 and O:3 showed low numbers of sublethally injured cells. C. sakazakii strains showed a higher survival rate after the exposure to drying (90 days) than other Cronobacter species. Strains (6 of 54) containing the thermotolerance genomic island tended to survive better at 58°C than other strains. In general, C. sakazakii strains were much more resistant than other Cronobacter species to environmental stresses such as desiccation, long-term drying and heat. This might explain why C. sakazakii strains are associated with PIF, milk powder and dry powdered foods, and more frequently isolated than other Cronobacter species. \n \nThis study analysed multiple methods for typing Cronobacter at the species and strain level, and showed different discriminatory powers. The present study recommended that using a combination of genomic cluster, chromogenic agar (DFI), and sialic acid, malonate, indole and inositol utilisation tests can be useful tools to distinguish the seven species of Cronobacter, distinguish pathogenic C. ST7 from other C. malonaticus STs, as well as to characterize and distinguish C. dublinensis strains to the subspecies level. The present study is an important contribution to the understanding of the diversity and characteristics of the Cronobacter genus using different typing methods, which is essential to reduce the risk of contaminations for the food products, in particular baby food such as PIF, milk powder and weaning food.