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

TL;DR

This paper presents the draft genomes of two Bacillus licheniformis strains from South African livestock that contain the lichenysin toxin operon.

Contribution

The novelty lies in the genome sequencing of B. licheniformis strains from goats and horses in South Africa, revealing the presence of the lichenysin operon.

Findings

The two strains have genome lengths of 4,152,826 and 4,110,075 bp.

Both genomes have a G + C content of 46%.

The lichenysin toxin operon was identified in both strains.

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%.

Tables

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

Full text

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% CO_2_. 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).

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 ASA^3^P 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 ASA^3^P (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.