ABSTRACT
Cronobacter is associated with infant infections and the consumption of reconstituted infant formula. Here we sequenced and closed six genomes of C. condimentiT, C. muytjensiiT, C. universalisT, C. malonaticusT, C. dublinensisT, and C. sakazakii that can be used as reference genomes in single nucleotide polymorphism (SNP)-based next-generation sequencing (NGS) analysis for source tracking investigations.
GENOME ANNOUNCEMENT
Cronobacter (formerly Enterobacter sakazakii) is a foodborne pathogen that has been identified as the causative agent of severe clinical complications in neonates and infants, such as meningitis, necrotizing enterocolitis, and septicemia (1, 2). The origin of this pathogen is not clear, but Cronobacter has been isolated from a wide range of foods, among which powdered infant formula (PIF) has been identified as the dominant vehicle of transmission (3, 4). Cronobacter is also often isolated from the environment and can be found in soil samples, domestic kitchens, and predominantly PIF manufacturing facilities (5, 6). The genus Cronobacter represents E. sakazakii, which was reclassified in 2007 as a result of biotyping and genotyping studies (7, 8).
Reliable identification and discrimination of Cronobacter strains is of importance due to the severe illness and ubiquitous occurrence in the environment and food. A multilocus sequence typing (MLST) scheme (9) has been shown to enable differentiation of closely related Cronobacter strains. The high discriminatory power and the drop in the cost of the next-generation sequencing (NGS) technologies favor the use of NGS as a routine diagnostic tool in public health reference laboratories in the near future (10). Clustering of Cronobacter isolates based on NGS data will allow a powerful source-tracking analysis. The clustering and the creation of phylogenic trees based on single nucleotide polymorphism (SNP) analysis of the NGS data are carried out by mapping short read sequences of Cronobacter isolates to a reference genome. The identification of reference genomes is essential for a reliable SNP-based analysis. Only a few complete genomes of Cronobater are available in public databases and the pool of reference genomes needs to be extended. Therefore, an effort was done to sequence and close genomes of Cronobacter spp. that can be used in SNP-based NGS analysis to support detailed source tracking investigations.
Genomic DNA was extracted from midexponential cultures using a Gentra DNA Purgene kit (Qiagen), and then 20-kb libraries were prepared following Pacific Biosciences (PacBio) protocol and Blupippin size selection. Sequencing was performed on the PacBio RSII platform using P4/C2 chemistry (P6/C4 for C. malonaticus) and three to four single-molecule real-time (SMRT) cells were used per strain with a 180-min (240 min for C. malonaticus) collection protocol. The subreads were de novo assembled using the PacBio Hierarchical Genome Assembly Process (HGAP)/Quiver software package (11), followed by minimus2 for genome circularization (12) and final polishing with Quiver. All the strains were assembled into a single contig corresponding to the chromosome. For some strains one to four circular plasmids were also obtained. The nucleotide sequences have been deposited at NCBI. The results of the sequencing and assemblies are summarized in Table 1. The genomes were annotated using the NCBI Prokaryotic Genomes Automatic Annotation Pipeline (PGAAP) and have been deposited at GenBank (NCBI).
Summary of genome sequencing and nucleotide accession numbers
During sequencing, epigenetic modifications of each nucleotide position were measured as kinetic variations (KVs) in nucleotide incorporation rates. Motifs were deduced from the KV data (13). Analysis were done using SMRT portal RS_Modification_and_Motif_Analysis Protocol.
Nucleotide sequence accession numbers.Sequences have been deposited in GenBank under the accession numbers listed in Table 1. Raw reads and motif summaries are deposited at SRA: C. condimentiT SRR2154341, C. muytjensiiT SRR2154340, C. sakazakii SRR2154342, C. universalisT SRR2154343, C. malonaticusT SRR3112550, and C. dublinensisT SRR2154345.
FOOTNOTES
- Received 8 February 2016.
- Accepted 9 February 2016.
- Published 24 March 2016.
- Copyright © 2016 Moine et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.
