Draft Genome Sequences of Bacillus licheniformis and Bacillus paralicheniformis Strains Isolated from Irish Skim Milk Powder

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.

B acillus 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)(2)(3)(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)(8)(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 280°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) highsensitivity (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 mL of each 2.0 nM normalized library. Subsequently, 600 mL 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. 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.