Complete Genome Sequences of Two Salmonella enterica subsp. enterica Serovar Anatum Strains Isolated from Papaya.

Salmonella enterica is a major global foodborne pathogen that causes gastroenteritis and, in some cases, death. Salmonella serovar Anatum has been increasingly associated with foodborne salmonellosis outbreaks. In this report, we announce two complete genome sequences of Salmonella Anatum isolated from papaya fruit.

ANNOUNCEMENT

Salmonella outbreaks linked to consumption of fresh papayas have caused over 350 reported cases of salmonellosis and 2 deaths in the United States since the first incidence in 2011 (www.cdc.gov/salmonella/). Salmonella enterica strains isolated from papaya are prevalent and highly diverse (1). A 2011 investigation by the U.S. Food and Drug Administration (FDA) isolated Salmonella spp. from 16% of tested papaya import samples from Mexico (n = 388), comprising isolates representing over 22 different serotypes (2). Many papaya-associated Salmonella isolates represent less-studied nontyphoidal serovars, highlighting a need for high-quality reference genomes for these strains. In this announcement, we report the complete genome sequences of two Salmonella enterica serovar Anatum papaya-associated isolates (CFSAN003959 and CFSAN003961).

CFSAN003959 was isolated from fresh papaya fruit sampled from a grower in Mexico in 2012. CFSAN003961 was isolated from fresh papaya fruit collected during import from Mexico to the United States in 2011. Collection of this sample occurred during increased sampling as a result of a 2011 Salmonella enterica serovar Agona outbreak. The Salmonella isolates were originally isolated and characterized as Salmonella as described in the Bacteriological Analytical Manual (BAM), chapter 5, “Salmonella” (https://www.fda.gov/food/laboratory-methods-food/bacteriological-analytical-manual-bam-chapter-5-salmonella).

Each isolate was cultured in Trypticase soy broth (Becton, Dickinson, Franklin Lakes, NJ, USA) overnight at 37°C. Genomic DNA was isolated from overnight cultures using the Promega Wizard Genomic DNA purification kit and Maxwell RSC Instrument (Promega Corporation, Madison, WI). Genomic DNA was sheared to approximately 20-kb fragments using a g-TUBE (Covaris, Inc., Woburn, MA). Sequencing libraries were prepared following the 20-kb PacBio sample preparation protocol using the BluePippin size selection system (Sage Science, Beverly, MA, USA). Each isolate was sequenced based on previously reported procedures on the PacBio RS II platform (Pacific Biosciences, Menlo Park, CA, USA) using two single-molecule real-time (SMRT) cells per isolate sequenced. Analysis of the sequence reads was implemented using SMRT Analysis 2.3.0. (https://smrt-analysis.readthedocs.io/en/latest/SMRT-Pipe-Reference-Guide-v2.3.0/). The continuous long-read data from each isolate were assembled de novo into one contig using the PacBio Hierarchal Genome Assembly Process 3 (HGAP3) program with default parameters. Gepard was used for identifying overlapping regions, and the closed genome sequences were rotated to start at the dnaA gene (3). Genomes were checked manually for even sequencing coverage. Then, the improved consensus sequence was uploaded in SMRT Analysis 2.3.0. to determine the final consensus and accuracy scores using the Quiver consensus algorithm. The sequencing statistics are listed in Table 1. The assembled sequences were annotated using the NCBI Prokaryotic Genome Annotation Pipeline and subsequently deposited at DDBJ/EMBL/GenBank (4).

TABLE 1

Sequence accession numbers and sequencing statistics

Nucleotide accession no.	Strain	No. of SMRT cells	SRA accession no.	Coverage (×)	Mean read length (bp)	No. of reads	N50 read length (bp)
CP041184	CFSAN003959	2	SRS622107	290	13,871	159,914	23,472
CP041183	CFSAN003961	2	SRS478508	260	12,108	137,193	21,206

The chromosome of Salmonella Anatum CFSAN003959 consists of 4,718,314 bp with a GC content of 52.6%. Three “intact” prophage regions were identified using PHASTER, including Salmonella phages Fels-1 and ST64T and prophage Gifsy-1 (5). One antimicrobial resistance gene, aac(6′)-Iaa, was identified using ResFinder (4). aac(6′)-Iaa is a chromosomally encoded aminoglycoside acetyltransferase that acetylates tobramycin, kanamycin, and amikacin (6).

The chromosome of Salmonella Anatum CFSAN003961 consists of 4,752,859 bp with a GC content of 52.6%. Four “intact” prophage regions were identified using PHASTER, including Salmonella phage Fels-1 and prophage Gifsy-1 (5). ResFinder analysis identified one antimicrobial resistance gene, aac(6′)-Iaa (7).

Data availability.

The complete genome sequences of Salmonella Anatum isolates CFSAN003959 and CFSAN003961 are publicly available in GenBank, and their details can be found in Table 1.