Complete Genome Sequence of Clostridium innocuum Strain ATCC 14501.

We report the complete genome sequence of Clostridium innocuum ATCC 14501, which was isolated in 1962 from an appendiceal abscess. At that time, the isolated strain was designated C. innocuum, given its suspected lack of virulence, but recent reports suggest that C. innocuum is an emerging pathogen.

ANNOUNCEMENT

We report the complete genome sequence of Clostridium innocuum strain ATCC 14501, which was identified in 1962 after isolation from an appendiceal abscess (1). It was characterized as a Gram-positive rod with terminal spores. Colonies were 1.5 to 2.5 mm in diameter, glossy, white, raised, and nonhemolytic. Intraperitoneal inoculation of the culture supernatant into mice was nonpathogenic. Since then, C. innocuum has been considered a benign gastrointestinal microorganism (1).

Now, the pathogenic potential of C. innocuum is being reconsidered. Recently, Chia and colleagues reported that C. innocuum is the second most common clostridial species causing extraintestinal infection (2) and is a cause of a Clostridioides difficile-like intestinal infection (3). These isolates were vancomycin resistant, cytotoxic to Vero and HT-29 cells, and enteropathogenic in mice (3). With this reconsideration of C. innocuum pathogenicity, the complete genome sequence of strain ATCC 14501 is needed.

Genomic DNA (gDNA) for both Illumina and Nanopore libraries was extracted using the BiOstic bacteremia DNA isolation kit (Qiagen, Germantown, MD) from an overnight culture grown anaerobically in tryptic soy broth at 37°C. Long-read sequencing libraries were prepared from unsheared gDNA using ligation sequencing kit SQK-LSK109 (Oxford Nanopore, UK) and sequenced on the MinION platform using a FLO-MIN106 flow cell. Default parameters were used for all software unless otherwise specified. Guppy v3.4.5 was used to base call reads with the R9.4.1 high-accuracy model and to perform read quality filtering based on Q scores, demultiplexing, and barcode trimming. Approximate read coverage was 98× from 19,646 reads totaling 460.0 Mbp. The read N50 value was 22,933 nucleotides, and the L50 value was 7,784 reads. Nanopore read quality was evaluated using pycoQC v2.5.0.21 (4).

Short-read sequencing libraries were prepared from gDNA using the Nextera XT kit (Illumina, San Diego, CA) and sequenced on the Illumina MiSeq platform using a v3 flow cell to yield 482,327 pairs of 301-bp reads. Sequencing adapters were trimmed from reads on-instrument to generate 196.3 Mbp of sequence, with an approximate genome coverage of 42×. Illumina read quality was evaluated using FastQC v0.11.2 (5). To minimize false-positive variant calls in alignments, no quality trimming of Illumina reads was performed (6).

The genome was assembled with Nanopore reads passing Q score filtering using Flye v2.6 to generate a single 4.30-Mb contig (7). Adapter-trimmed Illumina reads were aligned to the assembly using BWA v0.7.17, and assembly errors were corrected using Pilon v1.23 with a --mindepth setting of 0.1 (8, 9). Serial read alignment and Pilon correction were performed three times until no further assembly corrections were generated. Custom software (Pilon Tools v0.1) (https://github.com/egonozer/pilon_tools) was used to identify, manually assess, and correct any residual homopolymer assembly errors. Circulator v1.5.5 was used to ensure circularization of the chromosome by trimming overlapping ends and rotating to the start of the dnaA gene (10). The final assembly is a single chromosomal sequence with a length of 4,718,906 bp and a GC content of 43.6%. Annotation was performed using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) v4.11 (11, 12).

Data availability.

This project has been deposited in DDBJ/ENA/GenBank under accession number CP048838. The raw data accession numbers are SRR11552096 and SRR11552095.