ABSTRACT
Nineteen Bacillus licheniformis strains and four strains of the closely related species Bacillus paralicheniformis were isolated from a variety of Irish medium-heat skim milk powders. The draft genome sequences of these 23 isolates provide valuable genetic data for research work relevant to dairy products and process development. The isolates are available at Teagasc.
ANNOUNCEMENT
Bacillus licheniformis and Bacillus paralicheniformis are thermoresistant Gram-positive spore-forming bacteria that are commonly isolated from various dairy products, including milk powders, as well as farm environments (1–4). Neither species is clearly defined as pathogenic, but both have been associated with pathogenicity (5), food spoilage, and food poisoning incidents due to their toxin-producing ability.
B. licheniformis and B. paralicheniformis grow in a wide range of temperatures and facultative anaerobic conditions, making them difficult to control at all dairy processing steps (6).
In a previous screening study of the microbiota of Irish skim milk powders targeting spore-forming bacteria (4), 23 strains were isolated from different industrial sources. Those strains were obtained using one of two heat resistance treatments (80°C/10 min or 100°C/30 min), followed by incubation under aerobic or anaerobic conditions. Of the 23 strains, 11 were high heat-resistant spores that would be potentially challenging to inactivate during thermal processing technologies, such as high-temperature short-time (HTST) pasteurization, ultrahigh temperature (UHT) treatment, or spray drying (7–9). Enumeration, isolation, and purification of strains were performed as previously described (4).
The bacterial isolates are available at the Teagasc Food Safety Department (Moorepark) bacterial culture collection, where they are stored at −80°C.
For whole-genome sequencing, the isolates were incubated for 18 h at isolation temperature from a cryobead in 10 mL of brain heart infusion (BHI) broth. DNA was extracted from 2 mL of bacterial culture using the DNeasy UltraClean microbial kit (Qiagen, Venlo, Netherlands), following the manufacturer’s instructions, and quantified using a Qubit 2.0 fluorometer (Invitrogen, CA, USA), with the Qubit double-stranded DNA (dsDNA) high-sensitivity (HS) assay (Thermo Fisher Scientific), according to the supplier’s instructions. Library preparation and quantification were performed using the Nextera XT DNA sample preparation kit (Illumina, CA, USA) and the Qubit dsDNA HS assay, respectively. Library size distribution and quality were assessed using the 2200 TapeStation system (Agilent Technologies, CA, USA). Manual library normalization was performed, and the libraries were pooled with 5 μL of each 2.0 nM normalized library. Subsequently, 600 μL of a 12 pM library was made with a 1% PhiX control spike-in. Sequencing was performed on an Illumina MiSeq platform using the MiSeq reagent kit v3, yielding 300-bp paired-end reads.
Whole-genome sequencing analyses were performed using the TORMES v1.3.0 pipeline (10) with default software and parameters over the raw sequencing data, unless otherwise specified. The quality filtering process was performed using Trimmomatic v0.40 (11) (minimum read quality, 25; minimum read length, 50 bp). De novo genome assembly was carried out using SPAdes v3.15.2 (12) (minimum length of each contig to be kept in the genome after assembly, 200 bp). The quality and statistics of the assemblies were assessed using QUAST (13). Taxonomic identification of the sequencing data was performed using Kraken v2 (14). Genome annotation was conducted using Prokka v1.14.6 (15), and antimicrobial resistance screening was performed using BLASTN (16) built into ABRicate (Seemann; https://github.com/tseemann/abricate) and the ResFinder database (17).
The assembly statistics and main genome features are shown in Table 1.
TABLE 1.
General features of the genomes sequenceda
| Strain | Family (% of reads) | Genus (% of reads) | Species (% of reads) | No. of reads | No. of contigs (>200 bp) | Genome length (bp) | Largest contig (bp) | Contig N50 (bp) | GC content (%) | Sequencing depth (×) | No. of CDSs | No. of rRNAs | No. of tRNAs | No. of tmRNAs | Antimicrobial resistance screeninga |
GenBank accession no. | SRA accession no. | |||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Gene | Coverage (%) | % identity (GenBank accession no. of closest match) | Product | |||||||||||||||||
| DPTC10_3168b | Bacillaceae (98.89) | Bacillus (98.81) | Bacillus licheniformis (18.88) | 2,286,138 | 62 | 4,215,112 | 794,208 | 424,763 | 46.13 | 156 | 4,283 | 12 | 79 | 1 | JARAFA000000000 | SRR24425836 | ||||
| DPTC11_3245c | Bacillaceae (99.55) | Bacillus (99.49) | Bacillus licheniformis (14.87) | 1,380,580 | 70 | 4,191,660 | 1,288,030 | 303,315 | 46.16 | 96 | 4,262 | 10 | 81 | 1 | erm(D)_3 | 100 | 100 (M77505) | erm(D) | JARAFB000000000 | SRR24425835 |
| DPTC1_3328c | Bacillaceae (99.51) | Bacillus (99.45) | Bacillus licheniformis (14.06) | 3,087,310 | 683 | 4,625,408 | 566,540 | 420,093 | 45.99 | 191 | 4,575 | 12 | 79 | 1 | JARAFC000000000 | SRR24425824 | ||||
| DPTC13_3376c | Bacillaceae (99.52) | Bacillus (99.46) | Bacillus licheniformis (15.72) | 1,224,300 | 54 | 4,263,609 | 744,518 | 444,109 | 45.84 | 83 | 4,353 | 12 | 79 | 1 | JARAFD000000000 | SRR24425820 | ||||
| DPTC14_3226b | Bacillaceae (99.24) | Bacillus (99.18) | Bacillus paralicheniformis (90.52) | 2,166,970 | 58 | 4,437,393 | 782,344 | 478,415 | 45.6 | 141 | 4,408 | 12 | 81 | 1 | erm(D)_1 | 100 | 99.9 (M29832) | erm(D) | JARAFE000000000 | SRR24425819 |
| DPTC15_3285b | Bacillaceae (98.27) | Bacillus (98.19) | Bacillus paralicheniformis (88.19) | 2,409,736 | 91 | 4,527,348 | 844,392 | 242,197 | 45.8 | 151 | 4,542 | 17 | 82 | 1 | erm(D)_1 | 100 | 99.8 (M29832) | erm(D) | JARAFF000000000 | SRR24425818 |
| DPTC16_3330c | Bacillaceae (99.05) | Bacillus (98.96) | Bacillus paralicheniformis (90.51) | 5,371,688 | 56 | 4,454,351 | 900,159 | 620,291 | 45.65 | 346 | 4,427 | 12 | 80 | 1 | erm(D)_1 | 100 | 98.5 (M29832) | erm(D) | JARAFG000000000 | SRR24425817 |
| DPTC17_3130b | Bacillaceae (99.55) | Bacillus (99.49) | Bacillus licheniformis (18.73) | 2,272,542 | 93 | 4,360,113 | 605,381 | 368,842 | 45.83 | 152 | 4,467 | 11 | 79 | 1 | JARAFH000000000 | SRR24425816 | ||||
| DPTC18_3151b | Bacillaceae (99.56) | Bacillus (99.48) | Bacillus licheniformis (13.23) | 2,199,744 | 43 | 4,124,108 | 664,416 | 378,984 | 46.16 | 155 | 4,170 | 11 | 79 | 1 | JARAFI000000000 | SRR24425815 | ||||
| DPTC19_3196c | Bacillaceae (99.27) | Bacillus (99.17) | Bacillus licheniformis (20.59) | 3,415,084 | 46 | 4,264,904 | 1,295,074 | 989,612 | 45.83 | 232 | 4,360 | 12 | 79 | 1 | erm(D)_3 | 99.2 | 95.7 (M77505) | erm(D) | JARAFJ000000000 | SRR24425814 |
| DPTC20_3156c | Bacillaceae (99.00) | Bacillus (98.85) | Bacillus licheniformis (10.35) | 1,405,064 | 126 | 4,197,465 | 172,307 | 81,810 | 46.13 | 85 | 4,241 | 11 | 79 | 1 | JARAFK000000000 | SRR24425834 | ||||
| DPTC21_3181b | Bacillaceae (99.59) | Bacillus (99.53) | Bacillus licheniformis (15.20) | 2,582,824 | 65 | 4,154,884 | 1,288,031 | 303,300 | 46.22 | 182 | 4,203 | 10 | 79 | 1 | erm(D)_3 | 100 | 100 (M77505) | erm(D) | JARAFL000000000 | SRR24425833 |
| DPTC22_3213b | Bacillaceae (99.45) | Bacillus (99.38) | Bacillus licheniformis (13.30) | 2,636,076 | 68 | 4,186,141 | 1,278,173 | 511,798 | 46.16 | 181 | 4,254 | 12 | 79 | 1 | erm(D)_3 | 100 | 100 (M77505) | erm(D) | JARAFM000000000 | SRR24425832 |
| DPTC23_3165c | Bacillaceae (99.34) | Bacillus (99.26) | Bacillus licheniformis (18.45) | 6,270,052 | 163 | 4,417,735 | 757,750 | 383,392 | 45.77 | 407 | 4,488 | 12 | 80 | 1 | erm(D)_3 | 100 | 100 (M77505) | erm(D) | JARAFN000000000 | SRR24425831 |
| DPTC2_3370b | Bacillaceae (98.20) | Bacillus (98.12) | Bacillus paralicheniformis (89.26) | 2,540,672 | 1046 | 4,900,371 | 263,241 | 35,802 | 45.7 | 149 | 4,701 | 11 | 88 | 1 | erm(D)_3 | 93.9 | 98.5 (M77505) | erm(D) | JARAFO000000000 | SRR24425830 |
| DPTC24_3211c | Bacillaceae (99.32) | Bacillus (99.22) | Bacillus licheniformis (20.04) | 1,586,480 | 67 | 4,223,220 | 720,397 | 214,880 | 46.15 | 108 | 4,585 | 12 | 80 | 1 | JARAFP000000000 | SRR24425829 | ||||
| DPTC3_3122c | Bacillaceae (98.93) | Bacillus (98.85) | Bacillus licheniformis (19.36) | 3,297,568 | 126 | 4,271,439 | 1,076,265 | 438,762 | 46.29 | 224 | 4,348 | 11 | 79 | 1 | JARAFQ000000000 | SRR24425828 | ||||
| DPTC4_3301b | Bacillaceae (99.51) | Bacillus (99.44) | Bacillus licheniformis (25.93) | 2,711,002 | 48 | 4,623,884 | 1,317,640 | 405,549 | 45.31 | 170 | 4,819 | 11 | 81 | 1 | JARAFR000000000 | SRR24425827 | ||||
| DPTC5_3247b | Bacillaceae (95.88) | Bacillus (95.81) | Bacillus licheniformis (14.00) | 2,337,352 | 53 | 4,470,899 | 798,383 | 424,749 | 45.39 | 146 | 4,658 | 11 | 86 | 1 | JARAFS000000000 | SRR24425826 | ||||
| DPTC6_3313b | Bacillaceae (99.54) | Bacillus (99.47) | Bacillus licheniformis (19.87) | 1,974,030 | 98 | 4,451,921 | 604,659 | 252,913 | 45.73 | 127 | 4,606 | 12 | 80 | 1 | JARAFT000000000 | SRR24425825 | ||||
| DPTC7_3265c | Bacillaceae (99.21) | Bacillus (99.11) | Bacillus licheniformis (21.04) | 4,039,056 | 89 | 4,219,597 | 1,267,930 | 1,084,832 | 46.05 | 278 | 4,252 | 13 | 79 | 1 | erm(D)_3 | 99.2 | 95.7 (M77505) | erm(D) | JARAFU000000000 | SRR24425823 |
| DPTC8_3316c | Bacillaceae (98.98) | Bacillus (98.90) | Bacillus licheniformis (19.77) | 2,215,296 | 50 | 4,203,997 | 1,430,180 | 477,115 | 46.06 | 152 | 4,266 | 11 | 79 | 1 | erm(D)_3 | 99.2 | 95.7 (M77505) | erm(D) | JARAFV000000000 | SRR24425822 |
| DPTC9_3308b | Bacillaceae (98.80) | Bacillus (98.72) | Bacillus licheniformis (19.63) | 1,682,964 | 48 | 4,295,936 | 1,838,793 | 1,097,076 | 45.91 | 102 | 4,415 | 12 | 81 | 1 | JARAFW000000000 | SRR24425821 | ||||
The erm(D) gene confers resistance to erythromycin, lincomycin, clindamycin, quinupristin, pristinamycin IA, and virginiamycin S. Antimicrobial resistance screening of the genomes was conducted against ResFinder (17).
Spore heat resistance treatment of 80°C for 10 min.
Spore heat resistance treatment of 100°C for 30 min (highly heat-resistant spores).
Data availability.
The genome sequences of all 23 strains are publicly available at NCBI GenBank under BioProject accession number PRJNA913261 (“Spore-forming bacteria in skim milk powders”). The GenBank and Sequence Read Archive (SRA) accession numbers are listed in Table 1. This announcement represents the first version of all 23 genomes.
ACKNOWLEDGMENTS
We thank Evelyne Selberherr from the University of Veterinary Medicine, Vienna, for support with computing capabilities. This research was funded by the Department of Agriculture, Food and the Marine (DAFM) under the Food Institutional Research Measure (project number 14/F/883) and by Enterprise Ireland under the Dairy Processing Technology Centre (project number TC 2014 0016). A.L. is funded by Research Leaders 2025 postdoctoral funding from the European Union’s Horizon 2020 research and innovation program under Marie Skłodowska-Curie grant agreement number 754380. N.M.Q. is funded by the European Union’s Horizon 2020 research and innovation program under Marie Skłodowska-Curie grant agreement number 101034371.
Contributor Information
Antonio Lourenco, Email: Antonio.Lourenco@teagasc.ie.
Steven R. Gill, University of Rochester School of Medicine and Dentistry
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
The genome sequences of all 23 strains are publicly available at NCBI GenBank under BioProject accession number PRJNA913261 (“Spore-forming bacteria in skim milk powders”). The GenBank and Sequence Read Archive (SRA) accession numbers are listed in Table 1. This announcement represents the first version of all 23 genomes.