The complete genome sequence of a Shiga toxin-producing Escherichia coli (STEC) O26:H11 strain, MBT-5 (sequence type 21 [ST21], stx1a, stx2a, eae, ehxA), and two draft genome sequences of Listeria monocytogenes strains MBT-6 and MBT-7 belonging to the virulent sequence types 1 (ST1, clonal complex 1 [CC1]) and 59 (ST59, CC59), respectively, were determined. The strains were isolated in 2015 from ready-to-eat mixed greens in Germany.
ABSTRACT
The complete genome sequence of a Shiga toxin-producing Escherichia coli (STEC) O26:H11 strain, MBT-5 (sequence type 21 [ST21], stx1a, stx2a, eae, ehxA), and two draft genome sequences of Listeria monocytogenes strains MBT-6 and MBT-7 belonging to the virulent sequence types 1 (ST1, clonal complex 1 [CC1]) and 59 (ST59, CC59), respectively, were determined. The strains were isolated in 2015 from ready-to-eat mixed greens in Germany.
ANNOUNCEMENT
Shiga toxin-producing Escherichia coli (STEC) serotype O26 can lead to life-threatening infections and is the second most common serotype (after O157) found in clinical and food samples in Europe (1, 2). Listeria monocytogenes primarily afflicts immunocompromised individuals and can cause listeriosis with a mortality rate of about 20% (3). Both pathogens may enter the food chain through raw animal and vegetable products (4). The strains in this study were isolated by enrichment and using selective agar from fresh salad greens as previously described in detail (5). In addition to the species and serotyping confirmation done previously (5), here, the genome sequence data were determined to aid in virulence assessment.
Cultures were grown overnight at 37°C in brain heart infusion (BHI) broth, and genomic DNA was extracted using the ZR fungal/bacterial DNA miniprep kit (Zymo Research, Freiburg, Germany) for Listeria and the Genomic Micro AX Bacteria+ kit (A&A Biotechnology, Gdynia, Poland) for E. coli. The AX Bacteria+ kit is suitable for getting high-molecular-weight DNA. DNA quantification and paired-end (MiSeq; Illumina) and long-read (MinION; Oxford Nanopore) sequencing were performed as previously described (6). For short-read sequencing, the Nextera XT library kit and the MiSeq reagent kit v3 were used, and for long-read sequencing, the PCR barcoding genomic DNA (SQK-LSK109) protocol vNBE_9065_v109_revR_14Aug2019 with a MinION MK1B instrument was used. Default parameters were used for all software. The raw reads were quality controlled using FastQC v0.11.7 (https://github.com/s-andrews/FastQC). Hybrid assembly of short and long sequence reads of STEC strain MBT-5, obtained from Illumina and MinION sequencing, respectively, was performed with Unicycler v0.4.8 (7). The assembly of L. monocytogenes strains MBT-6 and MBT-7 was done with SPAdes v3.10.0 (8), with Illumina-generated sequences only. Annotation was done on the assembled contigs using the NCBI Prokaryotic Genome Annotation Pipeline v4.11 (9).
The hybrid assembly of STEC strain MBT-5 generated a single circular chromosome of 5,705,580 bp, with 6 circular plasmids (pMBT-5.1, 92,578 bp; pMBT-5.2, 89,589 bp; pMBT-5.3, 34,196 bp; pMBT-5.4, 6,715 bp; pMBT-5.5, 4,742 bp; pMBT-5.6, 2,784 bp). The circular chromosome of MBT-5 was rotated to dnaA as the origin with Geneious v9.2. Plasmids were not rotated. Strain MBT-5 was identified as serotype O26:H11, sequence type 21 (ST21), and adhesin fimH type 440 using SerotypeFinder v2.0, MLST v2.0, and FimTyper v1.0, respectively (10–12). Virulence genes were found using VirulenceFinder v2.0 (Table 1) (13). Interestingly, plasmid pMBT-5.1 harbored the virulence genes ehxA (enterohemolysin), espA (extracellular serine protease), toxB (toxin B), and katP (catalase peroxidase), while all other virulence genes were located on the chromosome.
TABLE 1.
Genome statistics of sequenced strains
| Strain | Platform (no. of raw reads; avg length [bp]) | Contig or no. of contigs | GC content (%) | Genome size (bp) | Contig type or N50 (bp) | Coverage (×) | No. of genes (CDS)a | ST (CC)b | Molecular Serotypec | Virulence genes determined by VirulenceFinder (total no.)d | GenBank accession no. | SRA accession no. |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Escherichia coli MBT-5 | Paired end using MiSeq (1,482,454; 251); MinION (127,117; 7,911) | Chromosome | 50.7 | 5,705,580 | Circular | 260 | 5,790 | ST25e | O26:H11 | astA, cif, eae, efa1, espB, espF, espJ, espP, fyuA, gad, iha, irp2, iss, iucC, iutA, lpfA, nleA, nleB, nleC, ompT stx1a, stx2a, terC, tir, tra (chromosome); ehxA, espA, katP, toxB (plasmid 5.1) (29) | CP058682 |
SRR12207194 (paired end), SRR12207193 (MinION) |
| Plasmid 5.1 | 47.8 | 92,578 | Circular | 281 | 93 | CP058683 | ||||||
| Plasmid 5.2 | 47.6 | 89,589 | Circular | 192 | 106 | CP058684 | ||||||
| Plasmid 5.3 | 39.7 | 34,196 | Circular | 386 | 36 | CP058685 | ||||||
| Plasmid 5.4 | 45.6 | 6,715 | Circular | 1,918 | 9 | CP058686 | ||||||
| Plasmid 5.5 | 41.2 | 4,742 | Circular | 1,151 | 4 | CP058687 | ||||||
| Plasmid 5.6 | 42.2 | 2,784 | Circular | 3,621 | 3 | CP058688 | ||||||
| Listeria monocytogenes MBT-6 | Paired end using MiSeq (669,662; 247) | 21 | 37.93 | 2,879,121 | 515,847 | 44 | 2,874 | ST1 (CC01) | 4b | agrA, flaA, flgC, flgE, gmar, lap, lapB, oatA, oppA, rli55, rli60, rsbv, bilE, bsh, chiA, clpB, clpc, clpe, clpp, codY, ctaP, ctsR, dal, degU, dltA, fbpA, fri, fur, hfq, hly, htrA, iap, inlA, inlB, inlC, inlH, inlJ, lgt, lhrC, lipA, lisR, lntA, lpeA, lplA1, lsp, mogR, mpl, mprf, murA, perR, pgdA, pgl, plcA, plcB, prfA, prsA2, pycA, recA, relA, secA2, sigB, sipX, sipZ, sod, srtA, srtB, stp, svpA, tcsA, tig, uhpT, virR (72) | JACBGM000000000 | SRR12207196 (paired end) |
| Listeria monocytogenes MBT-7 | Paired end using MiSeq (616,456; 247) | 24 | 37.83 | 2,975,327 | 482,161 | 45 | 2,985 | ST59 (CC59) | 1/2b | agrA, ami, aut, flaA, flgC, flgE, gmar, lap, lapB, oatA, oppA, rli55, rli60, rsbv, biLE, bsh, chiA, clpB, clpc, clpe, clpp, codY, ctaP, ctsR, dal, degU, dltA, fbpA, fri, fur, gtcA, hfq, hly, htrA, iap, inlA, inlB, inlC, inlH, inlJ, inlk, lgt, lhrC, lipA, lisR, lntA, lpeA, lplA1, lsp, mogR, mpl, mprf, murA, perR, pgdA, pgl, plcA, plcB, prfA, prsA2, pycA, recA, relA, secA2, sigB, sipX, sipZ, sod, srtA, srtB, stp, svpA, tcsA, tig, uHpt, virR (76) | JACBGL000000000 | SRR12207195 (paired end) |
The assembly of L. monocytogenes strains MBT-6 and MBT-7 generated 21 and 24 contigs, respectively. No plasmid sequences could be determined. Genome characteristics are shown in Table 1. The molecular serotype and multilocus sequence type (MLST) were identified using MLST 2.0 and multiplex PCR as described earlier (5). Both L. monocytogenes strains showed genetic characteristics similar to those of previously reported clinical, food, and outbreak-associated populations (ST1 = clonal complex 1 [CC1] and ST59 = CC59) (14), indicating that virulent STEC and L. monocytogenes strains can be transmitted via fresh produce. However, these strains could be isolated only at low incidence in northern Germany in 2015 (5).
Data availability.
The genome sequences have been deposited in GenBank under the BioProject number PRJNA643697. The sequences of STEC strain MBT-5 and L. monocytogenes strains MBT-6 and MBT-7 were deposited at DDBJ/ENA/GenBank under the accession numbers CP058682 (chromosome), CP058683 (plasmid 5.1), CP058684 (plasmid 5.2), CP058685 (plasmid 5.3), CP058686 (plasmid 5.4), CP058687 (plasmid 5.5), CP058688 (plasmid 5.6), JACBGM000000000, and JACBGL000000000. The raw sequencing data were also deposited at the SRA with the accession numbers shown in Table 1.
ACKNOWLEDGMENTS
We thank Adrian Prager, Gesa Gehrke, and Petra Horn for skillful technical assistance.
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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 have been deposited in GenBank under the BioProject number PRJNA643697. The sequences of STEC strain MBT-5 and L. monocytogenes strains MBT-6 and MBT-7 were deposited at DDBJ/ENA/GenBank under the accession numbers CP058682 (chromosome), CP058683 (plasmid 5.1), CP058684 (plasmid 5.2), CP058685 (plasmid 5.3), CP058686 (plasmid 5.4), CP058687 (plasmid 5.5), CP058688 (plasmid 5.6), JACBGM000000000, and JACBGL000000000. The raw sequencing data were also deposited at the SRA with the accession numbers shown in Table 1.