ABSTRACT
We report here the complete genome sequences of three Bacillus cereus group strains isolated from blood cultures from premature and immunocompromised infants hospitalized in intensive care units in three French hospitals. These complete genome sequences were obtained from a combination of Illumina HiSeq X Ten short reads and Oxford Nanopore MinION long reads.
ANNOUNCEMENT
The Bacillus cereus group comprises 18 closely related valid species, including B. cereus sensu stricto, that can cause extradigestive human infections, which are particularly severe in immunocompromised or premature infants (1–3). Since 2019, the literature on bacteremia in premature neonates has reported an increase in B. cereus infections. Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF) is widely used for bacterial identification, although its power of discrimination between closely related species is insufficient (4). Genome sequencing appears to be the best solution for proper identification. From 2013 to 2017, three strains identified as B. cereus based on MALDI-TOF were isolated from positive blood cultures from one immunocompromised infant and two premature infants hospitalized in intensive care units in three French hospitals. Colonies were obtained on 5% blood agar plates (Becton, Dickinson, Heidelberg, Germany) after incubation under an aerobic atmosphere at 35 ± 2°C for 24 h. Genomic DNA (gDNA) was extracted from pure colonies of the strains using a QIAamp DNA kit (Qiagen) according to the manufacturer’s instructions, with the addition of 100 μg/ml of lysozyme. Whole-genome sequencing (WGS) libraries were prepared according to the manufacturer’s protocols for Illumina libraries. Briefly, 1 μg genomic DNA was randomly fragmented and purified. The fragments were then end repaired, followed by A-tailing and ligation of the Illumina adaptors. After size selection, several rounds of PCR amplification were performed to enrich the adapter-ligated DNA fragments. After purification, the qualified libraries were sequenced in 150-bp paired-end format using the HiSeq X Ten platform. For Nanopore sequencing, the gDNA was not sheared, and size selection was performed using a BluePippin instrument (Sage Scientific) of 10 to 50 kb. The Nanopore libraries were generated according to the manufacturer’s instructions (genome DNA by ligation sequencing kit; SQK-LSK109). Sequencing was conducted using an R9.4.1 flow cell on a PromethION P48 instrument, and base calling was performed in real time using MinKNOW Core v3.6.8. An average read N50 value of 17,366 bp was obtained. The Illumina reads were trimmed using Trimmomatic v0.39 (5), and the final quality was manually checked using FastQ Screen v0.14 (6). The Nanopore reads were also filtered and trimmed using NanoLyse v1.2 and NanoFilt v2.6 (7), with a threshold quality score of 8. The sequencing depths for strains 18, 7, and 20 were, respectively, 17×, 20×, and 21× for Illumina and 86×, 24×, and 36× for Nanopore. The Illumina and Nanopore reads were then used as the input for a hybrid de novo genome assembly and circularization of the replicons using the Unicycler v04.9 pipeline (8) with default parameters. Manual finishing was performed based on similarity searches and synteny block detection using the Nanopore assembly sequence of each strain. Each of the 3 fully assembled genomes contains a single circular chromosome and plasmids (Table 1). They comprise, respectively, 5,746,647, 5,686,305, and 5,811,799 bases, with G+C contents of 35.3%, 35.5%, and 35.4%. Genome annotation was performed using the NCBI Prokaryotic Genome Annotation pipeline v5.1 (9). The similarity was assessed by genomic comparison with the sequences (DNA–DNA hybridization) of type strains using the Genome-to Genome Distance Calculator (GGDC) Web server (http://ggdc.dsmz.de/). This work reveals the misidentification of B. cereus group species by MALDI-TOF and shows that several species are responsible for bacteremia in preterm infants.
TABLE 1.
Genomic characteristics and accession numbers of the bacterial isolates Bacillus cereus strain 18, Bacillus luti strain 7, and Bacillus paranthracis strain 20a
| Characteristic | Data for strain: |
||
|---|---|---|---|
| 18 | 7 | 20 | |
| Bacterial speciesb | Bacillus cereus | Bacillus luti | Bacillus paranthracis |
| MLST | ST74 | ST24 | ST26 |
| Isolation date (yr/mo/day) | 2013/08/11 | 2017/03/31 | 2016/07/04 |
| Country: city | France: Nice | France: Paris | France: Marseille |
| BioSample accession no. | SAMEA8000806 | SAMEA6854266 | SAMEA6854265 |
| GenBank assembly accession no. | GCA_905221555 | GCA_905221595 | GCA_903545225 |
| GenBank accession no. | CAJNDP000000000 | CAJNDR000000000 | CAGVOV000000000 |
| Total no. of reads (ONT) | 118,533 | 258,028 | 252,077 |
| Total no. of reads (Illumina) | 9,041,928 | 8,831,560 | 8,955,974 |
| Genome size (bp) | 5,746,647 | 5,686,305 | 5,811,799 |
| Topology | Circular | Circular | Circular |
| No. of full genome sequences (chromosome + plasmids) | 1 + 6 | 1 + 3 | 1 + 4 |
| N50 (bp) | 5,318,884 | 5,469,117 | 5,275,862 |
| G+C content (%) | 35.3 | 35.5 | 35.4 |
| Total no. of genes | 5,911 | 5,761 | 5,963 |
| No. of protein-coding genes | 5,640 | 5,488 | 5,543 |
| No. of rRNAs | 42 | 42 | 42 |
| No. of 5S, 23S, and 16S rRNAs | 14 | 14 | 14 |
MLST, multilocus sequence type; ST, sequence type; ONT, Oxford Nanopore Technologies.
Strain identification by ≥96% similarity or ≥70% with digital DNA-DNA hybridization (dDDH; dDDH values of >70% are considered an indication that the tested organism belongs to a different species than the type strain[s] used as the reference[s]).
Data availability.
The three genome sequences and related metadata have been deposited in the GenBank database under accession numbers CAJNDP000000000, CAJNDR000000000, and CAGVOV000000000 for strains 18, 7, and 20, respectively. The raw reads were deposited in the SRA database under accession numbers SRR15372309, SRR15372310, and SRR15372308 (Illumina) and SRR15372306, SRR15372307, and SRR15372305 (Oxford Nanopore Technologies; ONT), respectively, for strains 18, 7, and 20.
ACKNOWLEDGMENTS
M.B.K. and F.N. benefited from a Ph.D. and a postdoc grant, respectively, from the Fondation Méditerranée Infection, Marseille, France. This work was financed by the French government through the UCAJEDI Investments in the Future project managed by the National Research Agency (ANR), with the reference number ANR-15-IDEX-01.
Contributor Information
Raymond Ruimy, Email: ruimy.r@chu-nice.fr.
Julie C. Dunning Hotopp, University of Maryland School of Medicine
REFERENCES
- 1.Bottone EJ. 2010. Bacillus cereus, a volatile human pathogen. Clin Microbiol Rev 23:382–398. doi: 10.1128/CMR.00073-09. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Glasset B, Herbin S, Granier SA, Cavalié L, Lafeuille E, Guérin C, Ruimy R, Casagrande-Magne F, Levast M, Chautemps N, Decousser J-W, Belotti L, Pelloux I, Robert J, Brisabois A, Ramarao N. 2018. Bacillus cereus, a serious cause of nosocomial infections: epidemiologic and genetic survey. PLoS One 13:e0194346. doi: 10.1371/journal.pone.0194346. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Lotte R, Hérissé A-L, Berrouane Y, Lotte L, Casagrande F, Landraud L, Herbin S, Ramarao N, Boyer L, Ruimy R. 2017. Virulence analysis of Bacillus cereus isolated after death of preterm neonates, Nice, France, 2013. Emerg Infect Dis 23:845–848. doi: 10.3201/eid2305.161788. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Manzulli V, Rondinone V, Buchicchio A, Serrecchia L, Cipolletta D, Fasanella A, Parisi A, Difato L, Iatarola M, Aceti A, Poppa E, Tolve F, Pace L, Petruzzi F, Rovere ID, Raele DA, Del Sambro L, Giangrossi L, Galante D. 2021. Discrimination of Bacillus cereus group members by MALDI-TOF mass spectrometry. Microorganisms 9:1202. doi: 10.3390/microorganisms9061202. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Bolger AM, Lohse M, Usadel B. 2014. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30:2114–2120. doi: 10.1093/bioinformatics/btu170. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Wingett SW, Andrews S. 2018. FastQ Screen: a tool for multi-genome mapping and quality control. F1000Res 7:1338. doi: 10.12688/f1000research.15931.2. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.De Coster W, D’Hert S, Schultz DT, Cruts M, Van Broeckhoven C. 2018. NanoPack: visualizing and processing long-read sequencing data. Bioinformatics 34:2666–2669. doi: 10.1093/bioinformatics/bty149. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Wick RR, Judd LM, Gorrie CL, Holt KE. 2017. Unicycler: resolving bacterial genome assemblies from short and long sequencing reads. PLoS Comput Biol 13:e1005595. doi: 10.1371/journal.pcbi.1005595. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Tatusova T, DiCuccio M, Badretdin A, Chetvernin V, Nawrocki EP, Zaslavsky L, Lomsadze A, Pruitt KD, Borodovsky M, Ostell J. 2016. NCBI prokaryotic genome annotation pipeline. Nucleic Acids Res 44:6614–6624. doi: 10.1093/nar/gkw569. [DOI] [PMC free article] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
The three genome sequences and related metadata have been deposited in the GenBank database under accession numbers CAJNDP000000000, CAJNDR000000000, and CAGVOV000000000 for strains 18, 7, and 20, respectively. The raw reads were deposited in the SRA database under accession numbers SRR15372309, SRR15372310, and SRR15372308 (Illumina) and SRR15372306, SRR15372307, and SRR15372305 (Oxford Nanopore Technologies; ONT), respectively, for strains 18, 7, and 20.