Complete Genome Sequences of 14 Salmonella enterica Serovar Enteritidis Strains Recovered from Human Clinical Cases between 1949 and 1995 in the United States.

Salmonella enterica serovar Enteritidis is one of the most commonly isolated foodborne pathogens and is transmitted primarily to humans through consumption of contaminated poultry and poultry products. We are reporting completely closed genome and plasmid sequences of historical S Enteritidis isolates recovered from humans between 1949 and 1995 in the United States.

GENOME ANNOUNCEMENT

An estimated 1.2 million people in the United States contract salmonellosis annually, resulting in 23,128 hospitalizations and >452 deaths (1). Since the 1980s, Salmonella enterica serovar Enteritidis has been one of the most common Salmonella serovars causing foodborne salmonellosis in the United States. S. Enteritidis is often transmitted to humans through contaminated eggs and other poultry products (2–6).

The lack of genetic variation within the serovar limits the use of conventional molecular subtyping methods for outbreak investigation and source attribution. Whole-genome sequencing is a viable platform for distinguishing outbreak isolates from epidemiologically distinct, but genetically related, S. Enteritidis strains (7). The availability of fully characterized and sequenced historical S. Enteritidis reference sequences significantly helps to increase our understanding of the evolution of this serovar. To facilitate this, we sequenced 14 S. Enteritidis isolates recovered from human clinical cases between 1949 and 1995 and released completely closed chromosomes and plasmids.

S. Enteritidis isolates were cultured in Trypticase soy broth (Becton Dickinson, Franklin Lakes, NJ, USA) at 37°C overnight. The genomic DNA was extracted using the DNeasy blood and tissue kit (Qiagen, Inc., Valencia, CA, USA) per the manufacturer’s protocol. The genomes were sequenced using the PacBio RS II sequencing platform, as previously reported (8). The library was prepared based on the 20-kb PacBio sample preparation protocol and sequenced using P6/C4 chemistry on four single-molecule real-time (SMRT) cells with a 240-min collection time. The continuous long-read (CLR) data were de novo assembled using the PacBio hierarchical genome assembly process (HGAP) (version 3.0) with default parameters (4). The assembled sequences were annotated using the NCBI Prokaryotic Genomes Annotation Pipeline v4 (https://www.ncbi.nlm.nih.gov/genome/annotation_prok/) and deposited at GenBank.

The S. Enteritidis genomes ranged between 4,661,885 and 4,934,512 bases and were fully closed, with coverage ranging between 119× and 517×. Multiple plasmids with sizes ranging between 33 kb and 244 kb were identified in some of the isolates. All plasmid sequences identified were completely closed. Resistome analysis was conducted using the publicly available resistance gene database, ResFinder, using a 90% sequence identity and 60% length minimum to identify acquired resistance genes (9). A summary of coverages, annotations, phenotypes, and resistomes is shown in Table 1.

TABLE 1 

Summary of genome sequence analysis and antimicrobial resistance genes identified in S. enteritidis

S. enteritidis strain	Yr of collection	GenBank accession no.	Chromosome/plasmid	Genome/plasmid size (bp)	Coverage (×)	Total no. of genes	Total no. of CDSa	No. of CDS excluding pseudogenes	No. of RNA genes	Resistance phenotypeb	Antimicrobial resistance genes identified
49-2444	1949	CP018633	Chromosome	4,661,885	149	4,846	4,726	4,519	120	Pan-susceptible
		CP018634	Plasmid pSE49-2444	92,891	101
56-3991	1956	CP018635	Chromosome	4,678,115	410	4,838	4,715	4,576	123	STR
		CP018636	Plasmid pSE56-3991	59,373	34
69-3861	1969	CP018637	Chromosome	4,690,881	423	4,994	4,874	4,709	120	AMP, FIS	blaOXA2, sul1
		CP018638	Plasmid pSE69-3861-1	95,773	126
		CP018639	Plasmid pSE69-3861-2	59,335	45
70-1605	1970	CP018640	Chromosome	4,679,538	478	4,832	4,709	4,570	123	STR
		CP018641	Plasmid pSE70-1605	59,371	48
74-1357	1974	CP018642	Chromosome	4,698,044	377	4,927	4,805	4,637	122	AMP, STR, TET	aph(3′')-Ib, aph(6)-Id, blaTEM-1, tetA
		CP018643	Plasmid pSE74-1357	118,824	1,409
77-2980	1977	CP018644	Chromosome	4,686,092	434	4,755	4,635	4,400	120	AMP, STR, TET
79-2359	1979	CP018645	Chromosome	4,684,908	270	4,827	4,703	4,499	124	AMP, STR	aph(6)-Id, aph(3′')-Ib, blaTEM-1
		CP018646	Plasmid pSE79-2359	54,796	50
81-1435	1981	CP018647	Chromosome	4,699,764	119	4,789	4,667	4,515	122	Pan-susceptible
81-1607	1981	CP018648	Chromosome	4,688,972	517	4,898	4,776	4,626	122	AMP, STR, TET, FIS	aph(6)-Id, aph(3′')-Ib, blaTEM-1
		CP018649	Plasmid pSE81-1607-1	54,541	202
		CP018650	Plasmid pSE81-1607-2	59,372	114
81-1705	1981	CP018651	Chromosome	4,698,445	424	5,152	5,022	4,835	130	AMP, CHL, STR, TET, FIS	aph(6)-Id, aph(3′')-Ib, blaTEM-1, cat1, tetD, tetC, tetB, sul2
		CP018652	Plasmid pSE81-1705-1	200,045	276
		CP018653	Plasmid pSE81-1705-2	59,334	36
		CP018654	Plasmid pSE81-1705-3	33,785	430
81-1706	1981	CP018655	Chromosome	4,699,800	307	5,091	4,969	4,753	122	AMP, CHL, STR, TET	aph(6)-Id, aph(3′')-Ib, blaTEM-1, cat1, tetB, tetC, tetD
		CP018656	Plasmid pSE81-1706	244,210	415
92-0392	1992	CP018657	Chromosome	4,934,512	286	5,202	5,082	4,900	120	AMP, STR, TET, FIS	aadA2, blacarB-2, sul1, tetG, floR
		CP018658	Plasmid pSE92-0392	94,039	86
93-0639	1993	CP018659	Chromosome	4,679,307	304	4,838	4,717	4,528	124	Pan-susceptible
		CP018660	Plasmid pSE93-0639	59,370	66
95-0621	1995	CP018661	Chromosome	4,679,624	221	4,893	4,771	4,593	122	AMP, TET	blaTEM-1, tetA
		CP018662	Plasmid pSE95-0621-1	59,356	28
		CP018663	Plasmid pSE95-0621-2	51,980	48

CDS, coding sequences.

AMP, ampicillin; CHL, chloramphenicol; STR, streptomycin; FIS, sulfisoxazole; TET, tetracycline.

Accession number(s).

The S. Enteritidis and plasmid sequences reported in this study have been deposited in the DDBJ/ENA/GenBank database under the accession numbers indicated in Table 1.