Complete Genome Sequences of Two Pasteurella multocida Isolates from Seabirds

Authors: Amro Hashish ⚬, Timothy J. Johnson, Dhiraj Chundru, Michele L. Williams, Yuko Sato, Nubia R. Macedo, Augustin Clessin, Hubert Gantelet, Caroline Bost, Jérémy Tornos, Amandine Gamble ⚬, Karen J. LeCount, Mostafa Ghanem, Thierry Boulinier, Mohamed El-Gazzar ⚬

Affiliations: a Department of Veterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine, Iowa State University, Ames, Iowa, USA; h National Laboratory for Veterinary Quality Control on Poultry Production, Animal Health Research Institute, Agriculture Research Center, Giza, Egypt; b Department of Veterinary and Biomedical Sciences, College of Veterinary Medicine, University of Minnesota, Minneapolis, Minnesota, USA; c Department of Veterinary Medicine, Virginia-Maryland College of Veterinary Medicine, University of Maryland, College Park, Maryland, USA; d CEFE, University of Montpellier, CNRS, EPHE, IRD, Montpellier, France; e Ceva Biovac, Beaucouzé, France; f University of California, Los Angeles, California, USA; g National Veterinary Services Laboratories, United States Department of Agriculture, Ames, Iowa, USA

License: Copyright © 2023 Hashish et al. CC BY 4.0 This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.

Article links: DOI: 10.1128/mra.01365-22  |  PubMed: 36971563  |  PMC: PMC10112064

Relevance: Moderate: mentioned 3+ times in text

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ANNOUNCEMENT

Recurrent outbreaks of fowl cholera (FC), caused by Pasteurella multocida, have been associated with mass die-offs; threatening the population of nestling albatrosses on Amsterdam Island, in the southern Indian Ocean (ref. 1, ref. 2, ref. 15, ref. 16, and ref. 17). Dead birds were necropsied on this island as part of a wildlife health monitoring program conducted since 2013 (ref. 1, ref. 3, ref. 4). The fieldwork was approved by the French Regional Animal Experimentation Ethical Committee no. 036 (Ministry of Research permit 10257-2018011712301381) and by the Comité de l’Environnement Polaire (A-2018-123 and A-2018-139 for 2018 to 2019). During necropsy, femur bone marrow was swabbed, and swabs were incubated in liquid Amies transport medium before cultivation (up to 8 h). The swabs were then inoculated on Columbia sheep blood and incubated at room temperature (13 to 20°C) for up to 48 h. Most abundant type colonies were selected and stored in stock culture agar at room temperature during transportation to the laboratory (up to several months). They were then streaked on blood medium plates and incubated at 37°C. Characterization of the obtained clonal populations was performed by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) (ref. 5). Isolates identified as P. multocida were stored at −80°C until sequencing. Two isolates of P. multocida—one from an adult yellow-nosed albatross (Thalassarche carteri) and one from an adult northern rockhopper penguin (Eudyptes moseleyi)—were sequenced in this study (Table 1).

Selected isolates were sequenced with the Illumina and Nanopore sequencing platforms for the purpose of hybrid assembly. The Circulomics Nanobind CBB Big DNA kit (Circulomics, Baltimore, MD, USA) was used for extraction of high-molecular-weight DNA for Nanopore sequencing. Nanopore libraries were prepared using the SQK-LSK109 and EXP-NBD104 kits according to the manufacturer’s protocol (Oxford Nanopore Technologies, United Kingdom). DNA shearing and size selection were not performed. Sequencing libraries were sequenced using MinION Mk1B sequencer (Oxford Nanopore Technologies) with R9.4.1 flow cells. For Illumina sequencing, bacterial DNA extraction was performed using MagMax pathogen RNA/DNA kit (Thermo Fisher Scientific, Waltham, MA, USA), as previously described (ref. 6), on a Kingfisher-Flex instrument (Thermo Fisher Scientific, Waltham, MA, USA). Subsequently, extracted DNA was used for the preparation of sequencing libraries using Illumina DNA Prep tagmentation kit (previously called Nextera DNA Flex) with IDT for Illumina DNA/RNA UD indexes and set A according to the manufacturer’s protocol (Illumina, USA). Sequencing was performed using the Illumina MiSeq system (v3 reagent kit using 2 × 300-bp paired-end reads).

Raw reads from Illumina data were trimmed for quality, and sequencing adapters were removed using Trimmomatic v.0.33 (ref. 7). For Nanopore sequencing, raw Nanopore electrical signal (.fast5) was converted to base sequence (.fastq) through high-accuracy basecalling using Guppy (v.4.2.3) within MinKNOW (20.10.6) software. NanoFilt version 2.8.0 was then used to quality-filter Nanopore reads and to filter out sequences less than 1,000 bp in length (ref. 8). Hybrid assemblies were performed using Unicycler v.0.5.0 (ref. 9). Default parameters were used for the software mentioned. Each of the sequenced genomes showed 2 circular contigs (1 chromosome and 1 plasmid). The genomes were annotated using NCBI Prokaryotic Genome Annotation Pipeline (PGAP) v.6.1 (ref. 10).

Sequencing, annotation, and typing results for the sequenced isolates are presented in Table 1. The availability of these complete genomes will help us to understand the source of P. multocida infections among seabirds through comparison to P. multocida genomes from other species.

Data availability.

The genomes are available from NCBI BioProject no, PRJNA839711 under the complete genome assembly accession no. (CP097610, CP097611, CP097612, and CP097613) shown in Table 1.

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