Draft genome sequences of Bacillus licheniformis strains MEZBL63 and MEZBL64 harboring the lichenysin toxin operon isolated from livestock in South Africa

ABSTRACT

Here, we report the draft genome sequences of two Bacillus licheniformis strains harboring the lichenysin operon that were isolated from healthy goat and horse in South Africa. The genomes were sequenced using Illumina MiSeq and had a length of 4,152,826 and 4,110,075 bp, respectively, with a G + C content of 46%.

ANNOUNCEMENT

Bacillus licheniformis is a ubiquitous, endospore-forming, Gram-positive saprophytic bacterium that belongs to the Bacillus subtilis group (1). It is an opportunistic pathogen in compromised hosts and it is characterized by high temperature and stress resistance (2). It causes a wide range of infrequent infections including gastroenteritis, bacteremia, peritonitis, and ophthalmitis in immunocompromised humans (3–6). It can cause toxemia, mastitis, and abortion in bovine (7, 8) and is implicated in foodborne poisoning and outbreaks (9–14). Lichenysin toxin-producing strains were isolated from raw milk and infant food products (8–10). Additionally, B. licheniformis has biotechnological applications (15), and it was used as a potential probiotic and antibiotics alternative to prevent pathogenic infection in broilers (16–18).

Here, we report draft genome sequences of B. licheniformis strains MEZBL63 and MEZBL64 isolated from fecal samples collected from a horse and a goat, respectively, in Flagstaff, OR Tambo District, Eastern Cape, South Africa in May 2018. The samples were collected as part of One Health research project investigating zoonotic diseases and antimicrobial resistance. The samples were collected in 10 mL of 0.1% buffered peptone water and were incubated for 24 h as reported previously (19). The samples were isolated using sheep blood agar for 18–24 h at 37°C in the presence of 5% CO₂. A single CFU was cultured in tryptic soy broth to an inoculum density of a 0.5-McFarland turbidity standard, and DNA was extracted from the cultures using DNeasy Blood & Tissue kit (Qiagen, Maryland, USA) as previously reported (19, 20). Sequencing libraries were prepared using Nextera XT Library Preparation Kit (Illumina Inc., California, USA). Sequencing was performed using Illumina MiSeq instrument (Illumina Inc., California, USA) and MiSeq Reagent Kit v2 (500 cycles). Sequencing yielded an average coverage of 29× and an average read length of 208 nt (Table 1).

TABLE 1.

Basic characteristics of the whole-genome sequencing, assemblies, and annotation of strains MEZBL63 and MEZBL64

Parameter	MEZBL63	MEZBL64
No. of raw reads	262,351	282,025
Average read length (nt)	220	219
Average coverage (×)	27.8	30.1
No. of contigs >200 bp	183	183
N50 (bp)	36,931	36,462
Genome size (bp)	4,152,826	4,110,075
G + C content (%)	46	46
No. of genes (total)	4,350	4,275
No. of CDSs (with protein)	4,204	4,118
No. of genes (RNA)	73	78
No. of rRNAs (5S, 16S, 23S)	1, 1, 1	1, 1, 1
Complete	1, 1, 1	1, 1, 1
Partial	0, 0, 0	0, 0, 0
No. of tRNAs	67	70
No. of ncRNAs	3	5
No. of pseudogenes (total)	73	79
Multilocus sequence type	3	3

Trimming (Trimmomatic v.0.36) (21), quality control (fastqc v.0.11.) (22), and assembly of raw data (SPAdes version 3.12.0) (23) were performed within the framework of ASA³P pipeline (v.1.4.0) (24). Contigs smaller than 200 nucleotides were discarded. Genome annotations were performed using NCBI Prokaryotic Genome Annotation Pipeline ( v.5.3) (25). The species were determined using TYGS pipeline (26). Sequencing, assembly, and annotation data of MEZBL63 and MEZBL64 are shown in Table 1. Assembly yielded a total length of 4,152,826 bp for MEZBL63 and 4,110,075 bp for MEZBL64, respectively. The GC content detected was 46%. An N₅₀ value of 36,931 bp (MEZBL63) and 36,462 bp (MEZBL64) was achieved.

Multilocus sequence type determination was performed using PubMLST database and the previously established genotyping scheme for B. licheniformis (27). Both MEZBL63 and MEZBL64 strains belong to sequence type 3.

Analysis of the genomes using ResFinder (v.4.1) (28) and PlasmidFinder (v2.1) (29) revealed that MEZBL63 and MEZBL64 harbored neither antibiotic resistance genes nor plasmids. Virulence genes were analyzed using VFDB database implemented in ASA³P (24), and no virulence genes were detected.

BLAST analysis using blast stand-alone version 2.10.0+ was performed in prfectblast v.2.0 (30) to determine the presence/absence of lichenysin operon (reference sequence: accession number GU949560.1) in both genomes. This analysis revealed that MEZBL63 and MEZBL64 harbored lichenysin operon (licABC/TE) albeit with several differences in the amino acid sequence, thus supporting the theory that they are two different isolates.

No other person had any role in the study concept, study design, data collection, experimental work, data analysis, data interpretation or the decision to publish. Any use of commercial names, commercial diagnostic products, or firm names is for descriptive purposes only and does not imply endorsement by the funding agencies, the National Institutes of Health, Food and Drug Administration, or the United States Government. Any opinions, findings and conclusions, or recommendations expressed in this material are those of the authors and do not necessarily reflect the view of the funders, the South Africa NRF, the US National Institutes of Health, or the United States Government. The funders of the study had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The corresponding author had full access to all the data in the study project and had the final responsibility for the decision to submit for publication.

DATA AVAILABILITY

https://www.ncbi.nlm.nih.gov/search/all/?term=PRJNA716986The whole-genome sequences were deposited at DDBJ/ENA/GenBank under the BioProject number PRJNA716986 (BioSample accession numbers SAMN23419540 and SAMN23419541, GenBank accession numbers JAJNNM000000000 and JAJNNL000000000 for MEZBL63 and MEZBL64, respectively. The versions described in this paper are the first versions. The sequences have been submitted to the Sequence Read Archive (SRA) under the accession numbers SRR17485928 and SRR17485927.