Fully Closed Genome Sequences of Five Type Strains of the Genus Cronobacter and One Cronobacter sakazakii Strain.

Cronobacteris associated with infant infections and the consumption of reconstituted infant formula. Here we sequenced and closed six genomes ofC. condimenti(T),C. muytjensii(T),C. universalis(T),C. malonaticus(T),C. dublinensis(T), andC. sakazakiithat 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).

TABLE 1

Summary of genome sequencing and nucleotide accession numbers

Organism	Chromosome size (bp)	No. of plasmids	Plasmid size (bp)	Accession no.
C. condimenti LMG 26250, CECT 7863T	4,366,820	1	164,790	CP012264 to CP012265
C. muytjensii ATCC 51329T	4,385,738	0	NAa	CP012268
C. sakazakii NCTC 8155	4,348,995	3	124,048/117,750/53,771	CP012253 to CP012256
C. universalis NCTC 9529T	4,323,715	1	136,454	CP012257 to CP012258
C. malonaticus LMG 23826, DSMZ 18702T	4,294,640	2	126,501/52,758	CP013940 to CP013942
C. dublinensis LMG 23823, DSMZ 18705T	4,444,709	1	203,534	CP012266 to CP012267

NA, not applicable.

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.