Draft genome sequence of Exiguobacterium sp. from whole cantaloupe, with inhibition capacity against Listeria monocytogenes

ABSTRACT We report the draft genome sequence of a novel species, Exiguobacterium sp., isolated from a freshly harvested and untreated cantaloupe in North Carolina. The strain Exiguobacterium wild type exhibited inhibitory activity against the foodborne pathogen Listeria monocytogenes, including strains of diverse serotypes and genotypes, both on agar media and in biofilms.

tion with fresh produce (3) makes Exiguobacterium spp.promising candidates as biocontrol agents for inhibition of the bacterial foodborne pathogen Listeria monocyto genes, implicated in produce-related outbreaks (4).A major outbreak of listeriosis in the USA in 2011 involved whole cantaloupe (4,5).Whole, ripe cantaloupe (Cucumis melo) was harvested from a field in North Carolina, USA (35°10′28″N, 77°48′44″W) and transported to North Carolina State University.Upon arrival at the laboratory, phosphatebuffered saline rinsates of the fruit were prepared and analyzed as described (6) for inhibition of L. monocytogenes 2011L-2858, derived from the 2011 cantaloupe outbreak (4), on soft (0.4%) trypticase soy (TS) agar (BD, Sparks, MD, USA).Zones of inhibition (~1.5 mm) were detected around light-yellow colonies of Gram-positive, rod-shaped bacteria, which were subsequently purified on TS agar.Analysis of 16S rRNA sequences using colony PCR and universal primers 8F and 1492R (7) indicated that the isolate is most closely related to Exiguobacterium acetylicum AMCC 101217 (CP030931.1)at 92% identity, and the strain was designated Exiguobacterium wild type (WT).Further tests done as described (6) showed that Exiguobacterium sp.WT inhibits growth (Fig. 1) of all tested strains of L. monocytogenes serotypes 1/2a, 1/2b and 4b, responsible for most human listeriosis (8).
Genomic DNA (gDNA) was extracted from Exiguobacterium WT grown in TS broth at 37°C overnight with the DNeasy Blood and Tissue kit (Qiagen, Valencia, CA, USA).The gDNA was quantified by a Qubit v.2.0 fluorometer and an Agilent 2200 TapeStation high molecular weight DNA assay.Paired-end libraries were prepared using 200 ng of gDNA with a TruSeq Nano DNA Library Prep (Illumina, San Diego, CA, USA).Briefly, the gDNA fragments, obtained using a Covaris S220 Ultrasonicator and purified using AMPure XP beads, were end-repaired followed by 550-bp insert size selection and enriched by PCR amplification.Quality and concentration of the amplified library was checked using an Agilent 2200 TapeStation (D1000) before sequencing on an Illumina NovaSeq 6000, utilizing a shared XP lane of 2 × 150-bp paired-end S4 flow cell reagent kit (Illumina).Data quality was checked using FastQC v.0.11.9 (9).Default parameters were used for all software unless otherwise specified.Genome assembly utilized SPAdes v.3.13.1 (10).
Annotation of genome assembly and putative pathways was done by the NCBI PGAP v.6.6 (11) (Table 1).Whole-genome sequence analysis indicated that the Exiguobacterium WT had the closest similarity to the plant-associated strains Leaf196 and RIT341 at 98.26% average nucleotide identity (ANI), and 91.12% ANI to the type strain E. acetylicum DSM20416.Considering the strain's inhibitory potential against L. monocytogenes, we subjected the genome to in silico secondary metabolite analysis using antiSMASH v.5.0 (12) and antibiotic resistance target seeker (ARTS v.2.0) (13).The genome harbored a putative biosynthetic pathway for the production of a toyoncin-like peptide active against the foodborne pathogens Bacillus cereus and L. monocytogenes (14).The genome contains two genes encoding L-alanyl-D-glutamate peptidase, an endolytic enzyme that degrades peptidoglycan and causes hydrolysis of the cell wall in L. monocytogenes and other bacteria (15).Exiguobacterium WT from cantaloupe fruit may represent a promising candidate for development of novel strategies to control L. monocytogenes in the food supply.

FIG 1
FIG 1 Inhibition zones produced by Exiguobacterium WT on lawns of L. monocytogenes.The inhibition zones are visible as dark rings around the spots of Exiguobacterium WT on the lawn of L. monocytogenes.The L. monocytogenes lawn is pale yellow, while the Exiguobacterium WT spots are darker yellow.Assays were done as described (6).Briefly, Exiguobacterium WT was spotted (5 µL) on soft (0.4%) TS agar mixed with L. monocytogenes 2011L-2858 (serotype 1/2b, panels A and B) and incubated at (A) 25°C, 48 h, and (B) 37°C, 24 h, or with L. monocytogenes F8027 (serotype 4b, panels C and D) and incubated at (C) 25°C, 48 h, and (D) 37°C, 24 h.L. monocytogenes and Exiguobacterium WT used for the lawns and the spots, respectively, were grown in TS broth for 24 h at the temperatures employed for the incubations.

TABLE 1
Assembly and annotation metrics