Draft Genome Sequences of 20 Clostridium botulinum Type A Isolates from Foodborne Botulism Outbreaks

ABSTRACT

Here, we present 20 draft genome sequences of Clostridium botulinum type A isolates originating from foodborne outbreaks in the United States and Ethiopia. Publicly available genomes enhance our understanding of C. botulinum genomics and are an asset in bioterrorism preparedness.

ANNOUNCEMENT

Clostridium botulinum produces botulinum neurotoxin (BoNT), which causes botulism, a severe and potentially life-threatening illness. The severity of the illness results in a need for surveillance of the organism and a greater understanding of its genomics (1). Here, we present 20 draft genome sequences of C. botulinum type A isolates that originated from clinical and food specimens from three unrelated foodborne botulism outbreaks: one that occurred in 2015 in Ethiopia due to butter made from powdered milk (outbreak 1) (2); one in the United States, due to home canned potatoes (outbreak 2) (3); and one in 2016, in the United States, due to “pruno,” an alcoholic beverage made in prisons (outbreak 3) (4, 5). Research work was done according to procedures approved by CDC’s Institutional Review Board (approval number 6911).

Strains were isolated using standard methods. Briefly, stool samples were streaked onto egg yolk agar, and single colonies were inoculated into both Trypticase peptone glucose starch (TPGY) and chopped meat glucose starch (CMGS) broth; each was incubated anaerobically at 37°C. After 5 to 7 days of growth, toxin testing was performed by mouse bioassay (6). Subsequently, all C. botulinum isolates were grown anaerobically in TPGY broth at 37°C for 16 to 18 h. Eight milliliters of culture was collected for DNA extraction, following a modified Epicentre protocol (7), as described previously (8). Samples from outbreaks 1 and 2 were prepared for sequencing on the Ion Torrent Personal Genome Machine (PGM); 200-bp libraries were created using the Kapa Biosystems library kit (9), pooled, and then enriched and amplified using the Ion ES and OneTouch2 systems (10). Chips were loaded manually with 16 pM of the pooled libraries and sequenced according to manufacturer’s instructions (11). Samples from outbreak 3 were prepared for sequencing on the Ion Torrent S5 system; 400-bp DNA libraries were created using the Kapa Biosystems kit (9), libraries were pooled, and amplification, enrichment, and chip loading were performed using the Ion Chef system, according to the manufacturer’s instructions (12). The chips were loaded with 200 pM of the pooled libraries and sequenced on the Ion Torrent S5 instrument, according to the manufacturer’s instructions (13).

Default settings were used on all analysis software unless otherwise specified. Base calling and read filtering to remove reads with an average Phred score below 27 were performed on the instrument using Torrent Suite v.4.6 (for outbreaks 1 and 2) (14) or Torrent Suite v.5.6.0 (for outbreak 3) (15). The reads were assessed using FastQC v.0.11.5 (16) and assembled using SPAdes v.3.13.0 (17), with –sc, –iontorrent, –careful, and -s flags enabled. The assemblies were assessed using QUAST v.5.0.0 (18) for contiguity, expected genome size, N₅₀ value, and GC content (Table 1) and filtered to remove contigs under 500 bp using the filter contigs script in CG-Pipeline (19). The predicted average coverage ranged from 20.1× to 433.9×; the expected genome size was 3.8 Mb for the samples from outbreaks 1 and 2 and 4.0 Mb for the samples from outbreak 3. The assembled genomes had a range of 63 to 216 contigs and an average GC content of 28.0%. Botulinum neurotoxin gene (bont) subtypes were determined using the “map reads to reference” tool in CLC Genomics Workbench v.11.0.0. Legacy 7-gene multilocus sequence typing (MLST) (20) types were determined by querying the PubMLST database for C. botulinum (21). The draft genomes were uploaded to GenBank and annotated using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) v.5.3 (22).

TABLE 1.

Sequencing results for the 20 isolates, along with accession numbers

Isolate ID	Country	State	Source	Source details	Final serotype	Toxin subtype	Sequencer	No. of reads	Avg read length (bp)	Avg coverage (×)	Seven-gene MLST STa	Total length (bp)	N50 (bp)	No. of contigs	GC (%)	BioSample accession no.	SRA accession no.	GenBank accession no.
CDC60066E1	Ethiopia	NAb	Clinical	NA	A	A2	PGM	777,446	164	31.9	99	3,819,818	47,974	151	28.0	SAMN17730278	SRR13605912	JANICI000000000
CDC60072E1	Ethiopia	NA	Food	Chili pepper paste	A	A2	PGM	707,815	168	29.7	99	3,803,210	46,362	170	28.0	SAMN17730280	SRR13605910	JANICH000000000
CDC60073B1	Ethiopia	NA	Food	Butter container	A	A2	PGM	647,265	219	35.4	99	3,830,021	116,170	89	28.1	SAMN17730281	SRR13605909	JANICG000000000
CDC60093T1	Ethiopia	NA	Food	Milk flakes	A	A2	PGM	651,458	185	30.1	99	3,815,871	34,727	193	28.0	SAMN17730283	SRR13605907	JANICF000000000
CDC60094PL1	Ethiopia	NA	Food	Milk flakes	A	A2	PGM	653,852	211	34.5	99	3,816,891	142,233	77	28.1	SAMN17730284	SRR13605906	JANICE000000000
CDC60143H1	USA	OH	Clinical	NA	A	A1	PGM	688,078	214	36.8	1	3,810,093	107,242	88	28.1	SAMN17850758	SRR13675311	JANICD000000000
CDC60148B1	USA	OH	Clinical	NA	A	A1	PGM	782,712	215	42.1	91	3,807,690	185,492	64	28.1	SAMN17850759	SRR13675310	JANICC000000000
CDC60150B1	USA	OH	Clinical	NA	A	A1	PGM	590,690	221	32.6	91	3,807,355	49,689	157	28.1	SAMN17850760	SRR13675309	JANIEW000000000
CDC60179	USA	OH	Clinical	NA	A	A1	PGM	421,935	196	20.7	1	3,812,548	45,882	160	28.0	SAMN17850762	SRR13675307	JANICB000000000
CDC60180	USA	OH	Clinical	NA	A	A1	PGM	549,139	189	25.9	1	3,807,697	56,932	128	28.0	SAMN17850763	SRR13675306	JANICA000000000
CDC60184	USA	OH	Food	Potato salad	A	A1	PGM	673,368	207	34.8	1	3,818,966	55,743	134	28.0	SAMN17850764	SRR13675305	JANIBZ000000000
CDC60185	USA	OH	Food	Potato salad	A	A1	PGM	477,504	206	24.6	1	3,805,620	146,502	63	28.0	SAMN17850765	SRR13675304	JANIBY000000000
CDC60186	USA	OH	Food	Potato salad	A	A1	PGM	526,375	198	26.1	1	3,810,226	71,267	123	28.0	SAMN17850766	SRR13675303	JANIBX000000000
CDC60191	USA	OH	Clinical	NA	A	A1	PGM	780,166	195	38.0	1	3,815,376	37,650	216	28.1	SAMN17850767	SRR13675302	JANIBW000000000
CDC61106H1	USA	MS	Clinical	NA	A	A1, B5	S5	1,901,893	311	147.9	6	4,020,025	96,592	107	27.9	SAMN17857382	SRR13676864	JANIBV000000000
CDC61108PL1	USA	MS	Clinical	NA	A	A1, B5	S5	1,466,086	309	113.3	6	4,037,871	117,613	86	27.9	SAMN17857383	SRR13676863	JANIBU000000000
CDC61111PL1	USA	MS	Clinical	NA	A	A1, B5	S5	5,509,915	315	433.9	6	4,022,266	139,874	107	27.9	SAMN17857384	SRR13676861	JANIBT000000000
CDC61113PL1	USA	MS	Clinical	NA	A	A1, B5	S5	2,114,503	332	175.5	6	4,033,440	76,976	125	27.9	SAMN17857386	SRR13676859	JANIBS000000000
CDC61118H1	USA	MS	Clinical	NA	A	A1, B5	S5	651,299	287	46.7	4	3,898,146	137,263	93	28.0	SAMN17857389	SRR13676856	JANIBR000000000
CDC61126T5	USA	MS	Clinical	NA	A	A1, B5	S5	2,099,563	280	147.0	6	4,040,446	102,517	85	27.9	SAMN17857390	SRR13676855	JANIBQ000000000

^aMLST, multilocus sequence type.

^bNA, not applicable.

Data availability.

The reads and genome sequences have been deposited at GenBank and the SRA as noted under BioProject accession number PRJNA428620.