# Draft Genome Sequence of Listeria innocua Strain MEZLIS29, Isolated from a Cow in South Africa

Mohamed E El Zowalaty, Alexandra Moura, Marc Lecuit, Oliver T Zishiri

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Mohamed E El Zowalaty, Alexandra Moura, Marc Lecuit, Oliver T Zishiri. Draft Genome Sequence of Listeria innocua Strain MEZLIS29, Isolated from a Cow in South Africa. Microbiology Resource Announcements, 2022, 11 (3), pp.e0112221. 10.1128/mra.01122-21. pasteur-03696087

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Submitted on 15 Jun 2022

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# Draft Genome Sequence of *Listeria innocua* Strain MEZLIS29, Isolated from a Cow in South Africa

© Mohamed E. El Zowalaty,ª © Alexandra Moura,ᵇ.c © Marc Lecuit,ᵇ.c,d © Oliver T. Zishiriᵉ

**ABSTRACT** Here, we report the draft genome sequence of *Listeria innocua* strain MEZLIS29, which was isolated from a healthy cow in Flagstaff, Eastern Cape Province, South Africa. The genome was sequenced using the Illumina MiSeq platform and had a length of 2,805,865 bp, with a G+C content of 37.5% and 2,783 coding DNA sequences, 58 tRNAs, 4 noncoding RNAs, and 8 rRNA genes.

Listeria species are small, motile, catalase-positive, non-spore-forming, rod-shaped, Gram-positive bacteria. The genus *Listeria* currently consists of 26 species (1), of which *Listeria monocytogenes* is a significant foodborne and invasive opportunistic pathogen causing serious epidemics and sporadic listeriosis in humans (2, 3). *Listeria innocua* is a nonpathogenic species that is closely related to *L. monocytogenes* (4). Although rare, atypical hemolytic *L. innocua* can actively cross the intestinal epithelium and spread systemically within the host (5). In addition to its ecological and clinical relevance (5–8), *L. innocua* genomes provide important insight into the evolution of pathogenicity of *L. monocytogenes* (4). Here, we report the draft genome sequence of *L. innocua* strain MEZLIS29, which was isolated from fecal material from a cow (*Bos taurus*) in Flagstaff, Eastern Cape, South Africa, in May 2018. The sample was analyzed as reported previously (9). The sample was collected in 10 mL of 0.1% buffered peptone water and incubated for 24 h. Following enrichment in *Listeria* broth (Oxoid, England), the sample was streaked onto *Listeria* selective agar (Oxoid) and incubated at 37°C for 18 h. A slant of the bacterial culture was shipped to the College of Veterinary Medicine, North Carolina State University, for further analysis. Colony PCR for the hemolysin (*hly*) gene was performed (10). An aliquot of overnight culture in brain heart infusion (BHI) broth was submitted to the Clinical Sciences Department at North Carolina State University for matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) using the VITEK MS identification system (bioMérieux, Marcy L'Etoile, France) and the associated software Knowledge Base v3.2 clinical US for further confirmation, as reported previously (9). The isolate was cultured on sheep blood agar for 18 to 24 h at 37°C in the presence of 5% CO₂, and a single CFU was cultured in tryptic soy broth (TSB) to an inoculum density of a 0.5 McFarland turbidity standard. An aliquot of the culture in TSB was diluted with 0.85% sterile saline solution to the desired inoculum density of 1 × 10⁶ CFU/mL using a Thermo Fisher Scientific Sensititre nephelometer, and the chilled culture tube was submitted to the University of Minnesota Genomics Center for DNA extraction and whole-genome sequencing (WGS). DNA isolation was performed using a Qiagen DNeasy blood and tissue kit (Lucigen, WI, USA) according to the manufacturer's protocol. The DNA sample was quantified using a fluorimetric PicoGreen assay using a Synergy 2 plate reader (BioTech). The purity of the DNA sample was also assessed using a Nanodrop 8000 instrument (Thermo Fisher).

**Editor** Steven R. Gill, University of Rochester School of Medicine and Dentistry

**Copyright** © 2022 El Zowalaty et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.

Address correspondence to Mohamed E. El Zowalaty, elzow005@gmail.com.

The authors declare no conflict of interest.

**Received** 18 November 2021

**Accepted** 10 January 2022

<footnote>ªZoonosis Science Center, Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden</footnote>

<footnote>ᵇInstitut Pasteur, National Reference Center and World Health Organization Collaborating Center for Listeria, Paris, France</footnote>

<footnote>ᶜInstitut Pasteur, Université de Paris, INSERM U1117, Biology of Infection Unit, Paris, France</footnote>

<footnote>ᵈNecker-Enfants Malades University Hospital, Division of Infectious Diseases and Tropical Medicine, APHP, Institut Imagine, Paris, France</footnote>

<footnote>ᵉDiscipline of Genetics, School of Life Sciences, College of Agriculture, Engineering, and Science, University of KwaZulu-Natal, Durban, South Africa</footnote>
<figure><img src="image_3.png" /><figcaption>**FIG 1** Positioning of isolate MEZLIS29 (highlighted in gray) within the *L. innocua* phylogeny. Representative genomes of *L. monocytogenes* were used as the outgroup. The maximum likelihood phylogeny was inferred from a core genome alignment of 632,995 nucleotide positions. Black circles represent bootstrap branch support values higher than 90%, based on 1,000 replicates.</figcaption></figure>

Sequencing libraries were prepared using a Nextera XT library preparation kit (Illumina Inc., CA, USA). Sequencing was performed using the MiSeq platform (Illumina Inc., CA, USA) using the v2 reagent kit, which yielded 250-bp paired-end (PE) reads. Sequence analyses were performed at the National Reference Center and World Health Organization Collaborating Center for Listeria (Institut Pasteur, Paris, France). Raw PE reads (251,655 reads [54× coverage]) were trimmed for adaptors and low-quality bases with AlienTrimmer (11). Cleaned reads (247,781 reads) were assembled using SPAdes v.3.12.0 (12) using the automatic k-mer, --only-assembler, and --careful options. Contigs smaller than 300 nucleotides were discarded. Assembly quality was assessed using QUAST v.2.2 (13), yielding a total of 73 contigs and 2,805,865 bp, with a G+C content of 37.5% and an N<sub>50</sub> value of 123,459 bp. The final sequence coverage of the assembled genome was 45×. The genome was annotated using the Prokaryotic Genome Annotation Pipeline (PGAP) v.5.1 (14) by the National Center for Biotechnology Information (NCBI), which identified 2,783 coding DNA sequences, 58 tRNAs, 4 noncoding RNAs, and 8 rRNA genes. A maximum likelihood phylogeny was inferred from the
core genome alignment of 43 *L. innocua* and 4 *L. monocytogenes* public genomes (5) using Parsnp, implemented in the Harvest suite v.1.1.2 (15), and was visualized with iTOL v.4.2 (16). Isolate MEZLIS29 clustered within clonal complex 537 (CC537) (nonhemolytic *L. innocua*) (Fig. 1) together with isolate MEZLIS26 (GenBank accession number AADHQU000000000), which was previously isolated from a goat in the same region (9). The average nucleotide identity by BLAST of MEZLIS29 and MEZLIS26 was 100%, confirming the circulation of the same strain in different animal species.

**Data availability.** The Whole Genome Shotgun project has been deposited in DDBJ/ENA/GenBank under BioProject accession number PRJNA716986 (BioSample accession number SAMN18492169) and GenBank accession number JAGISK0000000000. The version described in this paper is the first version, JAGISK0000000000.1. The sequences have been submitted to the Sequence Read Archive (SRA) under the accession numbers SRX10515072 and SRR14145944. All isolates used in this study are also publicly available at https://bigsdb.pasteur.fr/listeria.

## ACKNOWLEDGMENTS

We thank all funders who supported this work. The project was supported in part by the United States National Institutes of Health/Food and Drug Administration (FDA) under award 1U18FD006780-01, and the South African National Research Foundation supported this research through the Thuthuka Funding Instrument (grant TTK170411226583). The project was also supported in part by discretionary funds of M.E. El Zowalaty and O.T. Zishiri.

We thank staff members from the Veterinary Diagnostic Laboratory, College of Veterinary Medicine, and the Genomics Center at the University of Minnesota (St. Paul, MN, USA) for their cooperation and help throughout the project. We thank staff members from the Department of Population Health and Pathobiology, College of Veterinary Medicine, North Carolina State University (Raleigh, NC, USA), the GenomeTrakr Network, and the WGS Program for Foodborne Pathogen Traceback and the Center for Food Safety and Applied Nutrition (CFSAN), U.S. FDA, for their cooperation and support. We thank the NCBI GenBank submission staff for help with the genome uploading, decontamination, and deposition process.

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 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.

Mohamed E. El Zowalaty: Conceptualization, Data Curation, Formal Analysis, Investigation, Resources, Methodology, Validation, Visualization, Funding Acquisition, Writing – Original Draft, Review & Editing, Project Administration, Supervision. Alexandra Moura: Formal Analysis, Data Curation, Software, Investigation, Methodology, Visualization, Validation, Writing – Review & Editing. Marc Lecuit: Supervision, Project Administration, Software, Validation, Writing – Review & Editing. Oliver Zishiri: Resources, Supervision, Funding Acquisition, Project Administration, Writing – Review & Editing.

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