Abstract
BackgroundFollowing the association of Cronobacter spp. to several publicized fatal outbreaks in neonatal intensive care units of meningitis and necrotising enterocolitis, the World Health Organization (WHO) in 2004 requested the establishment of a molecular typing scheme to enable the international control of the organism. This paper presents the application of Next Generation Sequencing (NGS) to Cronobacter which has led to the establishment of the Cronobacter PubMLST genome and sequence definition database (http://pubmlst.org/cronobacter/) containing over 1000 isolates with metadata along with the recognition of specific clonal lineages linked to neonatal meningitis and adult infectionsResultsWhole genome sequencing and multilocus sequence typing (MLST) has supports the formal recognition of the genus Cronobacter composed of seven species to replace the former single species Enterobacter sakazakii. Applying the 7-loci MLST scheme to 1007 strains revealed 298 definable sequence types, yet only C. sakazakii clonal complex 4 (CC4) was principally associated with neonatal meningitis. This clonal lineage has been confirmed using ribosomal-MLST (51-loci) and whole genome-MLST (1865 loci) to analyse 107 whole genomes via the Cronobacter PubMLST database. This database has enabled the retrospective analysis of historic cases and outbreaks following re-identification of those strains.ConclusionsThe Cronobacter PubMLST database offers a central, open access, reliable sequence-based repository for researchers. It has the capacity to create new analysis schemes ‘on the fly’, and to integrate metadata (source, geographic distribution, clinical presentation). It is also expandable and adaptable to changes in taxonomy, and able to support the development of reliable detection methods of use to industry and regulatory authorities. Therefore it meets the WHO (2004) request for the establishment of a typing scheme for this emergent bacterial pathogen. Whole genome sequencing has additionally shown a range of potential virulence and environmental fitness traits which may account for the association of C. sakazakii CC4 pathogenicity, and propensity for neonatal CNS.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-1121) contains supplementary material, which is available to authorized users.
Highlights
Following the association of Cronobacter spp. to several publicized fatal outbreaks in neonatal intensive care units of meningitis and necrotising enterocolitis, the World Health Organization (WHO) in 2004 requested the establishment of a molecular typing scheme to enable the international control of the organism
Multilocus sequence typing of Cronobacter spp. and the Cronobacter PubMLST database The need for an internationally validated detection and molecular typing method for the genus Cronobacter is needed given the severe outcomes of infections in neonates and the attributed link to contaminated powdered infant formula on the international market [10,11,12]
The multilocus sequence typing (MLST) scheme covers all recognised species of Cronobacter genus, to better quantitate the intraspecific and interspecific diversity of the genus as well as potentially characterize the strains according to virulence groupings and source
Summary
Following the association of Cronobacter spp. to several publicized fatal outbreaks in neonatal intensive care units of meningitis and necrotising enterocolitis, the World Health Organization (WHO) in 2004 requested the establishment of a molecular typing scheme to enable the international control of the organism. Generation sequencing and multi-allelic profiling of emergent bacterial pathogens Sequence-based methods for bacterial identification started with single locus sequencing, such as the 16S rRNA gene. This can differentiate isolates between phylum to genus level and often to species level, but no further [1]. The application of multilocus sequence typing (MLST), typically 7 loci, enables the recognition of bacterial sequence types (STs) and clonal complexes (CCs). These loci were initially sequenced individually with designed primers. The use of whole genome MLST (>500 loci) to clone level is possible and has been applied in epidemiological studies for pathogen typing, e.g. Neisseria meningitidis, methicillin-resistant Staphylococcus aureus and bacterial population genomics of Campylobacter [3,4,5,6]
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