Complete genome sequences of Clostridium perfringens CP201 isolated from an asymptomatic rhesus monkey (Macaca mulatta)

ABSTRACT

Clostridium perfringens is a leading cause of human gastroenteritis. The genome sequence of C. perfringens CP201 from an asymptomatic rhesus monkey, Macaca mulatta, consists of one chromosome (3,241,413 bp; 30.22% G + C) and one plasmid (92,983 bp; 28.43% G + C), encoding 2,935 genes, 94 tRNAs, 30 rRNAs, and 1 CRISPR array.

ANNOUNCEMENT

Clostridium perfringens is the major pathogen causing gastroenteritis in humans and other animals worldwide (1, 2). Zoonotic diseases account for more than 60% of infections between humans and animals (3). Though extensive genome data of C. perfringens isolates from humans and poultry are currently available, those from nonhuman primates are rare (4, 5). Nonhuman primates provide unique biomedical information for bacterial infections because they share similarities to humans in genetics, physiology, anatomy, and immunology (3, 6, 7). The genome sequence of C. perfringens CP201 expands the current limited genome database in nonhuman primates and provides better understanding genetics of this human and animal pathogen.

Rhesus monkey fecal samples, collected from a nonhuman primate colony at a research center in Jefferson, Arkansas, were screened for anaerobic pathogens under the annual health monitoring program approved by the NCTR Institutional Animal Care and Use Committee. Diluted fecal samples (5 g feces in 45 mL 0.85% sterile saline) were directly streaked on TSA Blood agar (Remel, San Diego, CA) at 37°C for 2 days under anaerobic conditions (10% H₂, 5% CO₂, and 85% N₂). The pure culture of the isolate was obtained after successive culturing on TSA Blood agar in anaerobic conditions, The fast-growing Gram-positive rod-shaped isolate was identified as C. perfringens using a MALDI-TOF Biotyper (Bruker, Billerica, MA).

Genomic DNA was isolated from a single colony grown overnight under anaerobic conditions using the Blood & Tissue Kit (Qiagen, Redwood City, CA) and the quality and quantity of DNA were determined using NanoDrop spectrophotometer and Qubit fluorometer (Fisher, Waltham, MA). The same genomic DNA was used for Illumina and Nanopore sequencing without shearing or size selection. Following the manufacturer’s protocols for short reads, a genomic library was constructed using a Nextera XT Library Prep Kit and sequenced using MiSeq v2 Reagent Kit in the 250 bp paired-end mode on MiSeq (Illumina, San Diego, CA). Long-read sequencing was performed using the ligation sequencing kit SQK-LAK114, and flow cell FLO-MIN114 on Nanopore MinION: data acquisition and basecalling were done using MinKNOW 22.12.5 and Guppy 6.4.6 (Oxford Nanopore, Oxford, UK). Default parameters were used for all software unless otherwise noted. Raw short reads were quality checked and trimmed using CutAdapt 2.2 in BV-BRC^3.36.5 and raw long reads were processed using Porechop v0.2.1 and Trim Galore v0.6.7 in Galaxy (8). Short reads (n = 999,945) and long reads (n = 79,937; N₅₀, 2,593) were assembled with 127× depth using flye (2.91-b1780) in BV-BRC^3.36.5 (9) . The complete genome of C. perfringens CP201 is 3,334,396 bp in length, containing one chromosome (3,241,413 bp; 30.22% G + C) and one plasmid, pCP-P201 (92,983 bp; 28.43% G + C). The strain CP201 genome was annotated using the NCBI Prokaryotic Genome Annotation Pipeline v.6.6 (10). The annotated chromosome is predicted to have 2,935 coding genes, 41 pseudo genes, 94 tRNAs, 30 rRNAs, and 1 CRISPR array (Table 1). The close type genome is C. perfringens strain ATCC13124 (CP000246) with an ANI of 98.28%as estimated using MinHash v2.3 (11).

TABLE 1.

Summary of genetic features of C. perfringens strain CP201

Genetic element	Size (bp)	G + C content (%)	No. of coding sequences	No. of rRNAs	No. of tRNAs	GenBank accession no.
Chromosome	3,241,413	30.22	2,935	30	94	CP143271
Plasmid p4549-1	92,983	28.43	120	0	0	CP143272

DATA AVAILABILITY

The genome sequences have been deposited in GenBank under the accession numbers CP143271 and CP143272. The raw reads have been deposited in the Sequence Read Archive (SRA) under the accession numbers SRR27976716 and SRR27976717. The assembly has been deposited under the accession number GCA_020819895.2.