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Title: Diversity and virulence of the genus Cronobacter revealed by multilocus sequence typing (MLST) and comparative genomic analysis
Author: Joseph, S. M.
Awarding Body: Nottingham Trent University
Current Institution: Nottingham Trent University
Date of Award: 2013
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Cronobacter spp. (previously known as Enterobacter sakazakii) is a diverse bacterial genus consisting of opportunistic food-borne pathogens affecting all age groups, with particularly severe clinical complications such as meningitis and necrotising enterocolitis in neonates and infants. In this study, a multilocus sequence typing (MLST) approach has been established to span the entire Cronobacter genus, by employing the alleles of 7 housekeeping genes (atpD, fusA, glnS, gltB, gyrB, infB and ppsA, total length 3036 bp). The 325 Cronobacter spp. strains used in the study included isolates from the highly publicised Cronobacter cases from USA in December 2011. The scheme identified 115 sequence types (ST) across the seven Cronobacter species. Multilocus sequence analysis (MLSA) revealed considerable diversity in the genus, with intraspecific variation ranging from low diversity in C. sakazakii to extensive diversity within some species such as C. muytjensii and C. dublinensis including evidence of recombination events between species. An evolutionary analysis revealed the Cronobacter genus to have evolved 45-68 million years ago, during the period of evolution of flowering plants. The MLSA was also used in a polyphasic study for the formal recognition of two new species – C. universalis and C. condimenti. The MLST scheme also revealed the high level of clonality in the species C. sakazakii and C. malonaticus. ST4 was found to be a highly stable clone of C. sakazakii, and a strong association was established between the C. sakazakii ST4 clonal complex with neonatal meningitis cases. The curated MLST database is hosted with open access at: The diversity and virulence study of the organism was then extended to a whole genomic level by analysing eleven high quality draft Cronobacter spp. genomes spanning the seven species, including a representative of the C. sakazakii ST4 lineage, together with two publicly available genomes. Genome comparison revealed that pair-wise DNA sequence identity varies between 89 and 97% in the seven Cronobacter species. The number of annotated genes per genome varied between 3,700 and 4,200. The dataset revealed a pan-genome of more than 6000 genes, 32% of which was found to be conserved across the genus. Genes encoding adhesins, type six secretion systems, metal resistance genes as well as prophages were found in only subsets of genomes and have contributed considerably to the variation of genomic content. Sets of universal core genes and accessory genes unique to each species were identified, which can be used for designing genus/species specific detection assays. The C. sakazakii species was found to be unique in the Cronobacter genus in encoding genes enabling the utilization of exogenous sialic acid which may have considerable clinical significance. The C. sakazakii ST4 genome did not reveal any unique lineage-specific virulence related genes, suggesting the role of selective gene expression and increased host exposure in the pathogenicity of the organism.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available