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Complete genome sequences of eight Pasteurella multocida isolates representing all lipopolysaccharide outer core loci

Abstract

Pasteurella multocida (PM) is a major bacterial pathogen that causes fowl cholera disease in both domestic poultry and wild birds. Here, we report the complete genome sequences of eight PM isolates representing all known lipopolysaccharide outer core loci, which are phenotypically expressed as 16 known PM serotypes.

Article type: Brief Report

Keywords: Heddleston serotyping, somatic serotypes, lipopolysaccharide, complete genome sequence, hybrid assembly

Authors: Amro Hashish ⚬, Timothy J. Johnson, Mostafa Ghanem, Yuko Sato, Nubia R. Macedo, Karen J. LeCount, Mohamed El-Gazzar ⚬

Affiliations: Department of Veterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine, Iowa State University, Ames, Iowa, USA; National Laboratory for Veterinary Quality Control on Poultry Production, Animal Health Research Institute, Agriculture Research Center, Giza, Egypt; Department of Veterinary and Biomedical Sciences, College of Veterinary Medicine, University of Minnesota, Minneapolis, Minnesota, USA; Department of Veterinary Medicine, Virginia-Maryland College of Veterinary Medicine, University of Maryland, College Park, Maryland, USA; Diagnostic Bacteriology & Pathology Laboratory, National Veterinary Services Laboratories, U.S. Department of Agriculture, Animal and Plant Health Inspection Service, Veterinary Service, Ames, Iowa, USA

License: Copyright © 2024 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.00604-24  |  PubMed: 39365089  |  PMC: PMC11556135

Relevance: Moderate: mentioned 3+ times in text

Full text: PDF (219 KB)

ANNOUNCEMENT

Fowl cholera (FC), a disease caused by Pasteurella multocida (PM), is a contagious septicemic disease of domestic and wild birds associated with high morbidity and mortality (ref. 1). Industry reports continue to list FC as one of the top health problems the broiler, layer, and turkey industries are still facing (ref. 2).

PM isolates are differentiated into 16 somatic [lipopolysaccharide (LPS)] serotypes using Heddleston gel diffusion precipitin test (ref. 3, ref. 4). However, Heddleston serotyping has several limitations, including poor reproducibility, the inability to type many isolates, and cross-reactivity between serotypes (ref. 4ref. –ref. 6). Analysis of the LPS structure was conducted and showed that only eight unique LPS (L1–L8) outer core biosynthesis loci are found in the 16 Heddleston types (ref. 4). Subsequently, a multiplex PCR was developed to replace the Heddleston serotyping and type PM strains into one of the eight distinct LPS genotypes (ref. 4). Here, we report the complete genome sequences and annotation of eight PM isolates representing all eight LPS outer core biosynthesis loci. The availability of these genome sequences will promote the understanding of different PM LPS genotypes and offer new insight into prevention and control strategies of FC in all bird species.

Eight PM isolates were obtained from the Bacteriology Lab in the National Veterinary Services Laboratory. Bacterial isolation was performed from saved semi-solids (BD Diagnostic Systems, Sparks, MD, USA) of diagnostic and reference cultures. The cultures were plated onto blood agar (Remel, Lenexa, KS, USA) so that a single isolated colony could be picked and inoculated into fresh media. Then, bacterial DNA was extracted using MagMax (ref. 7). Both Illumina and Nanopore sequencing were conducted similar to our previous report (ref. 8). Extracted DNA was used for the preparation of sequencing libraries using the Illumina DNA Prep Tagmentation Kit (previously called Nextera DNA Flex) with IDT for Illumina DNA/RNA UD indexes and set according to the manufacturer’s protocol (Illumina, USA). Sequencing was performed using Illumina MiSeq system (v3 reagent kit using 2 × 300 bp paired-end reads). For nanopore sequencing, the Circulomics Nanobind CBB Big DNA Kit (Circulomics, Baltimore, MD, USA) was used for the extraction of high molecular weight DNA. Libraries were prepared using SQK-LSK109 and EXP-NBD104 kits according to the manufacturer’s protocol (Oxford Nanopore Technologies, UK).

Short reads generated using Illumina were trimmed for quality and sequencing adapters using Trimmomatic version 0.33, using default parameters (ref. 9). NanoFilt version 2.8.0 was used with default parameters to quality filter Nanopore reads and filter for sequences less than 1,000 bp in length (ref. 10). Hybrid assemblies were performed using Unicycler version 0.5.0 using default parameters (ref. 11). The presence or absence of plasmid from the genome sequences was determined using the Plasmid database (ref. 12) available via PLSDB https://ccb-microbe.cs.uni-saarland.de/plsdb/.

All of the genomes were annotated using the Pathosystems Resource Integration Center (PATRIC) (ref. 13) genome annotation tool, which adopts the RAST tool kit (RASTtk) (ref. 14).

A summary of the data associated with the eight sequenced PM strains, reads generated by each sequencing platform, assembly statistics, annotation of the closed genomes, sequence typing, average nucleotide identity, and GenBank accession numbers are presented in Table 1.

TABLE 1: Data associated with the eight sequenced PM strains, reads generated by each sequencing platform, assembly statistics, annotation of the closed genomes, sequence typing, average nucleotide identity, and GenBank accession numbers

Parameter Data
Metadata PM-10159 PM-1702 PM-10957 PM-2192 PM-2095 PM-2237 PM-1581 PM-2723
Host Turkey Turkey Turkey Turkey Turkey Turkey Pine Siskin Turkey
Geographical location USA-Minnesota USA-Virginia USA-Arkansas USA-Texas USA-Minnesota USA-Iowa USA- Massachusetts USA-Indiana
Date of sample collection 3/3/2015 Unknown 8/28/2019 Unknown Unknown Unknown Unknown Unknown
Raw sequence reads Illumina paired-end read length (nt) 300 250 300 250 250 250 250 250
Number of Illumina reads used 244,732 973,588 210,138 868,126 1,056,934 1,437,582 1,319,566 1,393,248
Average Illumina coverage 32× 104× 27× 96× 113× 151× 146× 152×
Number of Nanopore reads 2,300 29,033 19,162 58,979 15,947 29,000 27,322 16,492
Average Nanopore coverage 14× 176× 147× 350× 94× 161× 192× 108×
Nanopore reads N50(bp) 33,748 34,507 40,787 36,346 34,937 29,770 39,271 36,641
Assembly statistics for the closed genomes No. of contigs 1 2 1 2 2 1 2 2
Total length of the chromosome (bp) 2,277,732 2,337,700 2,314,161 2,268,200 2,338,462 2,379,541 2,253,720 2,297,391
Contig matching a plasmid Absent Present Absent Present Present Absent Present Present
Total length of the plasmid NA 2,516 NA 3,346 3,682 NA 1,812 2,507
GC content (%) 40.48 40.44 40.35 40.43 40.33 40.20 40.47 40.37
N50 for the hybrid assembly (bp) 2,277,732 2,337,700 2,314,161 2,268,200 2,338,462 2,379,541 2,253,720 2,297,391
Annotation results Number of coding sequences 2,128 2,273 2,126 2,116 2,226 2,260 2,102 2,211
Number of tRNAs 58 56 57 57 60 55 57 57
Number of rRNAs 19 19 19 19 19 19 19 19
Hypothetical proteins 273 301 261 264 323 326 253 284
Proteins with functional assignments 1,854 1,972 1,865 1,852 1,903 1,934 1,849 1,927
Proteins with Enzyme Commission numbers assignments (ref. 15) 714 768 726 720 737 722 723 759
Proteins with Gene Ontology assignments (ref. 16) 559 604 572 568 578 568 570 600
Proteins with Pathway assignments (ref. 17) 496 534 508 499 513 499 505 524
Proteins with PATRIC genus-specific family (PLfam) assignments (ref. 18) 2,101 2,232 2,081 2,058 2,182 2,217 2,030 2,155
Proteins with PATRIC cross-genus family (PGfam) assignments (ref. 18) 2,103 2,235 2,090 2,072 2,184 2,221 2,039 2,168
DNA fingerprinting Restriction endonuclease analysis, digestion with HhaI (ref. 19) 1,014 0005 1,023 0006 0009 0015 0008 0016
Serotyping Using gel diffusion precipitin test (GDPT) (ref. 19) 1 5 4 6 9 15 8 16
ANIb (%)T1_FN1(cut-off threshold of 96%) (ref. 20) To the PM type strain (NCTC10322) LT906458.1 96.28 98.47 98.39 97.99 98.31 98.17 96.08 98.42
Genotypes Lipopolysaccharide genotypeT1_FN2 L1 L2 L3 L4 L5 L6 L7 L8
GenBank data BioSample accession number SAMN28548360 SAMN28553018 SAMN28552779 SAMN28553023 SAMN28553020 SAMN28553350 SAMN28552999 SAMN28553054
BioProject accession number PRJNA839711 PRJNA839711 PRJNA839711 PRJNA839711 PRJNA839711 PRJNA839711 PRJNA839711 PRJNA839711
SRA (MinION)SRA (Illumina) SRX15391466SRX15391459 SRX15391475SRX15391487 SRX15391465SRX15391458 SRX15391477SRX15391461 SRX15391476SRX15391460 SRX15391479SRX15391463 SRX15391473SRX15391485 SRX15391478SRX15391462
Complete genome assembly accession number Chromosome CP097604 Chromosome CP097616 ChromosomeCP097606 ChromosomeCP097620 ChromosomeCP097618 ChromosomeCP117196 ChromosomeCP097614 ChromosomeCP097622
Plasmid CP097617 PlasmidCP097621 PlasmidCP097619 PlasmidCP097615 PlasmidCP097623

Average nucleotide identity percentage was calculated based on BLAST+ (21) available via JSpeciesWS online service https://jspecies.ribohost.com/jspeciesws/#home.

Lipopolysaccharide genotyping according to Harper et al. (4).

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