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).
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.
Footnotes
Citation Moine D, Kassam M, Baert L, Tang Y, Barretto C, Ngom Bru C, Klijn A, Descombes P. 2016. Fully closed genome sequences of five type strains of the genus Cronobacter and one Cronobacter sakazakii strain. Genome Announc 4(2):e00142-16. doi:10.1128/genomeA.00142-16.
REFERENCES
- 1.Healy B, Cooney S, O’Brien S, Iversen C, Whyte P, Nally J, Callanan JJ, Fanning S. 2010. Cronobacter (Enterobacter sakazakii): an opportunistic foodborne pathogen. Foodborne Pathog Dis 7:339–350. doi: 10.1089/fpd.2009.0379. [DOI] [PubMed] [Google Scholar]
- 2.Jaradat ZW, Al Mousa W, Elbetieha A, Al Nabulsi A, Tall BD. 2014. Cronobacter spp.—opportunistic foodborne pathogens. A review of their virulence and environmental-adaptive traits. J Med Microbiol 63:1023–1037. doi: 10.1099/jmm.0.073742-0. [DOI] [PubMed] [Google Scholar]
- 3.Jaradat ZW, Ababneh QO, Saadoun IM, Samara NA, Rashdan AM. 2009. Isolation of Cronobacter spp. (formerly Enterobacter sakazakii) from infant food, herbs and environmental samples and the subsequent identification and confirmation of the isolates using biochemical, chromogenic assays, PCR and 16S rRNA sequencing. BMC Microbiol 9:225. doi: 10.1186/1471-2180-9-225. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Pan Z, Cui J, Lyu G, Du X, Qin L, Guo Y, Xu B, Li W, Cui Z, Zhao C. 2014. Isolation and molecular typing of Cronobacter spp. in commercial powdered infant formula and follow-up formula. Foodborne Pathog Dis 11:456–461. doi: 10.1089/fpd.2013.1691. [DOI] [PubMed] [Google Scholar]
- 5.Craven HM, McAuley CM, Duffy LL, Fegan N. 2010. Distribution, prevalence and persistence of Cronobacter (Enterobacter sakazakii) in the nonprocessing and processing environments of five milk powder factories. J Appl Microbiol 109:1044–1052. doi: 10.1111/j.1365-2672.2010.04733.x. [DOI] [PubMed] [Google Scholar]
- 6.Molloy C, Cagney C, O’Brien S, Iversen C, Fanning S, Duffy G. 2009. Surveillance and characterisation by pulsed-field gel electrophoresis of Cronobacter spp. in farming and domestic environments, food production animals and retail foods. Int J Food Microbiol 136:198–203. doi: 10.1016/j.ijfoodmicro.2009.07.007. [DOI] [PubMed] [Google Scholar]
- 7.Iversen C, Lehner A, Mullane N, Bidlas E, Cleenwerck I, Marugg J, Fanning S, Stephan R, Joosten H. 2007. The taxonomy of Enterobacter sakazakii: proposal of a new genus Cronobacter gen. nov. and descriptions of Cronobacter sakazakii comb. nov. Cronobacter sakazakii subsp. sakazakii, comb. nov., Cronobacter sakazakii subsp. malonaticus subsp. nov., Cronobacter turicensis sp. nov., Cronobacter muytjensii sp. nov., Cronobacter dublinensis sp. nov. and Cronobacter genomospecies 1. BMC Evol Biol 7:64. doi: 10.1186/1471-2148-7-64. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Joseph S, Cetinkaya E, Drahovska H, Levican A, Figueras MJ, Forsythe SJ. 2011. Cronobacter condimenti sp. nov., isolated from spiced meat, and Cronobacter universalis sp. nov., a species designation for Cronobacter sp. genomospecies 1, recovered from a leg infection, water and food ingredients. Int J Syst Evol Microbiol 62:1277–1283. [DOI] [PubMed] [Google Scholar]
- 9.Baldwin A, Loughlin M, Caubilla-Barron J, Kucerova E, Manning G, Dowson C, Forsythe S. 2009. Multilocus sequence typing of Cronobacter sakazakii and Cronobacter malonaticus reveals stable clonal structures. BMC Microbiol 9:223. doi: 10.1186/1471-2180-9-223. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Köser C, Ellington M, Cartwright E, Gillespie S, Brown N, Farrington M, Holden M, Dougan G, Bentley S, Parkhill J, Peacock S. 2012. Routine use of microbial whole genome sequencing in diagnostic and public health microbiology. PLoS Pathog 8:e1002824. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Chin CS, Alexander DH, Marks P, Klammer AA, Drake J, Heiner C, Clum A, Copeland A, Huddleston J, Eichler EE, Turner SW, Korlach J. 2013. Nonhybrid, finished microbial genome assemblies from long-read SMRT sequencing data. Nat Methods 10:563–569. doi: 10.1038/nmeth.2474. [DOI] [PubMed] [Google Scholar]
- 12.Sommer DD, Delcher AL, Salzberg SL, Pop M. 2007. Minimus: a fast, lightweight genome assembler. BMC Bioinformatics 8:64. doi: 10.1186/1471-2105-8-64. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Korlach J, Turner SW. 2012. Going beyond five bases in DNA sequencing. Curr Opin Struct Biol 22:251–261. doi: 10.1016/j.sbi.2012.04.002. [DOI] [PubMed] [Google Scholar]