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Microbiol Resour Announc
Microbiol Resour Announc
mra
Microbiology Resource Announcements
2576-098X
American Society for Microbiology 1752 N St., N.W., Washington, DC

39162446
mra00244-24
10.1128/mra.00244-24
mra.00244-24
Genome Sequences
veterinary-microbiologyVeterinary MicrobiologyGenome sequences of Clostridium perfringens isolated from diseased dogs
https://orcid.org/0000-0002-2524-9713
Sekse Camilla 1 Conceptualization Funding acquisition Investigation Methodology Project administration Supervision Writing – original draft camilla.sekse@vetinst.no

Solsvik Helene K. 2 Investigation Methodology Resources Writing – review and editing
Haverkamp Thomas H. A. 1 Formal analysis Methodology Writing – review and editing
https://orcid.org/0000-0002-9559-1303
Kaspersen Håkon 1 Data curation Formal analysis Visualization Writing – review and editing
Gulliksen Wenche S. 3 Formal analysis Writing – review and editing
Campos Sabrina R. 2 Funding acquisition Investigation Methodology Resources Supervision Writing – review and editing
Nørstebø Simen F. 2 Investigation Methodology Resources Supervision Writing – review and editing
Bøe Cathrine A. 3 Conceptualization Methodology Supervision Writing – review and editing
1 Department of Animal Health and Food Safety, Norwegian Veterinary Institute , Ås, Norway
2 Bacteriology and Mycology Unit, Department of Paraclinical Sciences, Faculty of Veterinary Medicine, Norwegian University of Life Sciences , Ås, Norway
3 Department of Analysis and Diagnostics, Norwegian Veterinary Institute , Ås, Norway
Editor Putonti Catherine Loyola University Chicago , Chicago, Illinois, USA

Address correspondence to Camilla Sekse, camilla.sekse@vetinst.no
The authors declare no conflict of interest.

9 2024
20 8 2024
20 8 2024
13 9 e00244-2413 3 2024
05 7 2024
Copyright © 2024 Sekse et al.
2024
Sekse et al.
https://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.

ABSTRACT

Two Clostridium perfringens isolates originating from two Norwegian dogs with acute hemorrhagic diarrhea were sequenced. Based on Illumina and Oxford Nanopore Technology sequencing, hybrid assemblies were generated, and one of the genomes was completed and closed. For both isolates, virulence genes and their genomic location have been identified.

KEYWORDS

Clostridium perfringens
diseased dogs
acute hemorrhagic diarrhea
Norwegian Veterinary Institute SEQ-TECH Sekse Camilla Haverkamp Thomas H. A. Kaspersen Håkon Gulliksen Wenche S. Bøe Cathrine A. cover-dateSeptember 2024
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pmcANNOUNCEMENT

In the autumn of 2019, there was an outbreak of acute hemorrhagic diarrhea (AHD) in dogs in Norway. Providencia alcalifaciens was pointed out as a likely cause of the outbreak with a possible secondary role of Clostridium perfringens (1). C. perfringens can harbor a range of different toxins, among which the NetF-toxin has particularly been associated with AHD in dogs (2, 3) as well as netE, netG, and cpe (4–6).

We aimed to characterize two C. perfringens genomes from two dogs presenting with AHD during the outbreak. Here, we report hybrid assemblies of C. perfringens based on Illumina and Oxford Nanopore Technologies (ONTs), along with the identification and localization of virulence genes.

Presumptive C. perfringens were isolated from stool samples after inoculation onto a blood agar plate incubated anaerobically at 37°C for 18–24 h and then verified with MALDI Biotyper MS (MALDI-TOF MS, Daltonics GmbH) (1). DNA was extracted from an overnight culture from a brain heart infusion medium. For ONT sequencing, the Gentra PureGene Yeast/Bact. A kit (QIAGEN) was used, following the supplier’s protocol for Gram-positive bacteria. For Illumina sequencing, the QIAamp DNA Mini Kit (QIAGEN) with minor changes to the protocol was used. DNA concentrations were determined using the Qubit dsDNA BR Assay Kit (Thermo Fisher Scientific), and DNA quality was assessed using the MySpec spectrophotometer (VWR). Libraries for Illumina sequencing were made using the Illumina DNA prep (Illumina) followed by sequencing on NextSeq 500 (Illumina) with 150 bp paired-end chemistry. High-quality DNA (~400 ng) from each sample was prepared for ONT sequencing using a Rapid Barcoding library preparation kit (SQK-RBK004, ONT). Pooled libraries were cleaned using AMPure XP beads (Beckman Coulter). The barcoded library (10 µL) was sequenced in two successive rounds on FLO-FLG001 flow cells on a MinION device (ONT) for ~24 h. Raw ONT sequence data were basecalled separately after each run using Guppy (v.6.5.7; www.nanoporetech.com), with the basecalling model dna_r9.4.1_450bps_sup.cfg and a minimum quality score of 7. Basecalled sequences were demultiplexed using qcat (v.1.1.0; https://github.com/nanoporetech/qcat), and sequence quality was checked with Nanoplot (v.1.33.1) (7). Default parameters were used for all software unless otherwise specified.

Filtlong (v.0.2.1; https://github.com/rrwick/Filtlong) was used to filter out the lowest quality 10% of the Nanopore reads and discard any reads shorter than 1,000 bp. Unicycler (8) (v.0.5.0) was used to generate hybrid assemblies based on ONT and Illumina reads, either with the normal (2019–01-3486-1) or bold (2019–01-3502-1) mode. Polypolish (9) (v.0.5.0) was used to correct the hybrid assemblies. Quast (10) (v.5.2.0) was run to determine the quality of the assemblies, followed by coverage calculations with BWA (11) (v.0.7.8), SamTools (12) (v.1.3.1), and BedTools (13) (v.2.31.0).

BLAST (v.2.13.0) was used to run a nucleotide blast search against a local database of virulence genes of interest (4, 14), using default parameters.

The genome of C. perfringens 2019–01-3486-1 was completed by a closed chromosome and six plasmids. C. perfringens 2019–01-3502-1 is a draft genome containing several smaller contigs, in combination with a presumably completed chromosome and plasmids (Table 1), in total 33 contigs. A description of genomes including virulence genes is presented in Table 1.

TABLE 1 Overview of Clostridium perfringens genomes including plasmids and virulence characteristics

ID	# Tot. reads
(Illumina, 150 bp)	Accession number
(Illumina fastq)	# Tot. reads
(ONT, N50)	# contigs	Accession number
(Nanopore fast5)	Size (Mbp)	GC (%)	Coveragea	Biosample, Accession number	
2019–01-3486-1	4,319,645	SRR25822369	29,724
(9,131 bp)	7	SRR25896867	3.64	28.17	352.70	SAMN37194955	
2019–01-3502-1	1,957,084	SRR25822368	59,184
(2,839 bp)	33	SRR25896866	3.71	28.23	156.80	SAMN37194956	
ID	Replicon ID	N contigs	Sum contig size (bp)	Virulence genes	Replicon accession	
 2019–01-3486-1	1	1	3,357,566	pfoA, cpa/plc	CP134262	
	2	1	74,370		CP134263	
	3	1	72,367	netE, netF	CP134264	
	4	1	66,544		CP134265	
	5	1	48,498	cpe	CP134266	
	6	1	13,415		CP134267	
	7	1	3,332		CP134268	
 2019–01-3502-1	1	1	3,532,600	pfoA, cpa/plc	CP134229	
	2	1	72,750	netE, netF	CP134230	
	5	1	13,553		CP134233	
	6	1	4,410		CP134234	
	3, 4, 7–33b	29	82,768	netG, cpe	NDc	
a Based on short reads only.

b Incomplete contigs.

c No accession number for these contigs as they are not closed plasmids/chromosome.

ACKNOWLEDGMENTS

This work was part of SEQ-TECH, an internal, strategic investment project from the Norwegian Veterinary Institute (2019–2023). H.K.S. was supported by the research track program for veterinary students at the Faculty of Veterinary Medicine, Norwegian University of Life Sciences.

The bioinformatics work was performed on resources provided by UNINETT Sigma2 - the National Infrastructure for High Performance Computing and Data Storage in Norway.

Thanks to the veterinarians and the dog owners who supported the investigation of the outbreak in 2019 and the bacteriology laboratory at the Norwegian Veterinary Institute for clinical bacteriology examination of the samples. The samples were obtained with an owner consent, which includes a general clause providing permission to use the samples and its secondary material for research.

DATA AVAILABILITY

This whole-genome sequencing project has been deposited to ENA under accession number PRJNA1010682, BioSample SAMN37194955 and SAMN37194956 (Table 1).
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REFERENCES

1 Jørgensen HJ, Valheim M, Sekse C, Bergsjø BA, Wisløff H, Nørstebø SF, Skancke E, Lagesen K, Haaland AH, Rodriguez-Campos S, Sjurseth SK, Hofshagen M, Jarp J, Tronerud O-H, Johannessen GS, Heggelund M, Rygg S, Christensen E, Boye M, Gjerset B, Sandvik M, Soltvedt EM, Wolff C. 2021. An official outbreak investigation of acute haemorrhagic diarrhoea in dogs in Norway points to Providencia alcalifaciens as a likely cause. Animals (Basel) 11 :3201. doi:10.3390/ani11113201 34827932
2 Sindern N, Suchodolski JS, Leutenegger CM, Mehdizadeh Gohari I, Prescott JF, Proksch A-L, Mueller RS, Busch K, Unterer S. 2019. Prevalence of Clostridium perfringens netE and netF toxin genes in the feces of dogs with acute hemorrhagic diarrhea syndrome. J Vet Intern Med 33 :100–105. doi:10.1111/jvim.15361 30499621
3 Leipig-Rudolph M, Busch K, Prescott JF, Mehdizadeh Gohari I, Leutenegger CM, Hermanns W, Wolf G, Hartmann K, Verspohl J, Unterer S. 2018. Intestinal lesions in dogs with acute hemorrhagic diarrhea syndrome associated with netF-positive Clostridium perfringens type A. J Vet Diagn Invest 30 :495–503. doi:10.1177/1040638718766983 29621942
4 Mehdizadeh Gohari I, Kropinski AM, Weese SJ, Whitehead AE, Parreira VR, Boerlin P, Prescott JF. 2017. NetF-producing Clostridium perfringens: clonality and plasmid pathogenicity loci analysis. Infect Genet Evol 49 :32–38. doi:10.1016/j.meegid.2016.12.028 28062388
5 Mehdizadeh Gohari I, Parreira VR, Nowell VJ, Nicholson VM, Oliphant K, Prescott JF. 2015. A novel pore-forming toxin in type A Clostridium perfringens is associated with both fatal canine hemorrhagic gastroenteritis and fatal foal necrotizing enterocolitis. PLOS ONE 10 :e0122684. doi:10.1371/journal.pone.0122684 25853427
6 Mehdizadeh Gohari I, Kropinski AM, Weese SJ, Parreira VR, Whitehead AE, Boerlin P, Prescott JF. 2016. Plasmid characterization and chromosome analysis of two netF+ Clostridium perfringens isolates associated with foal and canine necrotizing enteritis. PLoS ONE 11 :e0148344. doi:10.1371/journal.pone.0148344 26859667
7 De Coster W, D’Hert S, Schultz DT, Cruts M, Van Broeckhoven C. 2018. NanoPack: visualizing and processing long-read sequencing data. Bioinformatics 34 :2666–2669. doi:10.1093/bioinformatics/bty149 29547981
8 Wick RR, Judd LM, Gorrie CL, Holt KE. 2017. Unicycler: resolving bacterial genome assemblies from short and long sequencing reads. PLoS Comput Biol 13 :e1005595. doi:10.1371/journal.pcbi.1005595 28594827
9 Wick RR, Holt KE. 2022. Polypolish: short-read polishing of long-read bacterial genome assemblies. PLoS Comput Biol 18 :e1009802. doi:10.1371/journal.pcbi.1009802 35073327
10 Mikheenko A, Prjibelski A, Saveliev V, Antipov D, Gurevich A. 2018. Versatile genome assembly evaluation with QUAST-LG. Bioinformatics 34 :i142–i150. doi:10.1093/bioinformatics/bty266 29949969
11 Li H, Durbin R. 2009. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25 :1754–1760. doi:10.1093/bioinformatics/btp324 19451168
12 Danecek P, Bonfield JK, Liddle J, Marshall J, Ohan V, Pollard MO, Whitwham A, Keane T, McCarthy SA, Davies RM, Li H. 2021. Twelve years of SAMtools and BCFtools. Gigascience 10 :giab008. doi:10.1093/gigascience/giab008 33590861
13 Quinlan AR, Hall IM. 2010. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26 :841–842. doi:10.1093/bioinformatics/btq033 20110278
14 Liu B, Zheng D, Zhou S, Chen L, Yang J. 2022. VFDB 2022: a general classification scheme for bacterial virulence factors. Nucleic Acids Res 50 :D912–D917. doi:10.1093/nar/gkab1107 34850947
