Genetic diversity of Salmonella enterica isolated over 13 years from raw California almonds and from an almond orchard

Abstract

A comparative genomic analysis was conducted for 171 Salmonella isolates recovered from raw inshell almonds and raw almond kernels between 2001 and 2013 and for 30 Salmonella Enteritidis phage type (PT) 30 isolates recovered between 2001 and 2006 from a 2001 salmonellosis outbreak-associated almond orchard. Whole genome sequencing was used to measure the genetic distance among isolates by single nucleotide polymorphism (SNP) analyses and to predict the presence of plasmid DNA and of antimicrobial resistance (AMR) and virulence genes. Isolates were classified by serovars with Parsnp, a fast core-genome multi aligner, before being analyzed with the CFSAN SNP Pipeline (U.S. Food and Drug Administration Center for Food Safety and Applied Nutrition). Genetically similar (≤18 SNPs) Salmonella isolates were identified among several serovars isolated years apart. Almond isolates of Salmonella Montevideo (2001 to 2013) and Salmonella Newport (2003 to 2010) differed by ≤9 SNPs. Salmonella Enteritidis PT 30 isolated between 2001 and 2013 from survey, orchard, outbreak, and clinical samples differed by ≤18 SNPs. One to seven plasmids were found in 106 (62%) of the Salmonella isolates. Of the 27 plasmid families that were identified, IncFII and IncFIB plasmids were the most predominant. AMR genes were identified in 16 (9%) of the survey isolates and were plasmid encoded in 11 of 16 cases; 12 isolates (7%) had putative resistance to at least one antibiotic in three or more drug classes. A total of 303 virulence genes were detected among the assembled genomes; a plasmid that harbored a combination of pef, rck, and spv virulence genes was identified in 23% of the isolates. These data provide evidence of long-term survival (years) of Salmonella in agricultural environments.

Fig 1. Maximum likelihood tree based on SNPs identified by aligning 171 de novo assemblies to the reference chromosome of Salmonella Typhimurium LT2 ASM694v2 with Parsnp and genotypic antimicrobial resistance (AMR).

The scale bar is in number of SNPs. The colors in the phylogenetic tree represent different serogroups; squares represent a chromosomal location and circles represent a plasmid location for AMR genes. AMR genes are color-coded by antibiotic classes.

Fig 2. Phylogenetic tree of Salmonella Enteritidis generated with the CFSAN SNP Pipeline.

The branch lengths represent the SNP distances among the isolates. Almond survey isolates (black text) and outbreak-associated orchard isolates (green dot) were compared to clinical isolates (red text) from the 2001 and 2006 outbreaks (retrieved from the NCBI database). Almond isolates of Salmonella Enteritidis PT 9c (LJH1024) and Salmonella Enteritidis PT 30 (LJH0608) from the 2004 and 2001 outbreaks, respectively, were included for comparison.

Fig 3. Phylogenetic tree of Salmonella Montevideo generated with the CFSAN SNP Pipeline.

The branch lengths represent the SNP distances among the isolates.

Fig 4. Phylogenetic tree of Salmonella Typhimurium and 1,4,[5],12:i:-.

The branch lengths are representative of the SNP distances among the isolates.

Fig 5. General features of plasmids identified in Salmonella survey isolates included in this study.

(A) Putative plasmids predicted were categorized as mobilizable, conjugative, or non-mobilizable based on the presence or absence of relaxase, mate-pair formation marker genes and/or oriT sequence. Each colored circle represents a plasmid-carrying Salmonella isolate. (B) Frequency of plasmid replication types.

Fig 6. Heatmap of the distribution of plasmid replication types detected in Salmonella isolates.

Fig 7. Heatmap of the distribution of virulence genes encoding type III secretion system (T3SS) across 171 genomes.

The shades of gray represent the number of open reading frames (ORF) that are detected in each putative virulence gene.

Fig 8. Heatmap of the distribution of genes encoding the fimbrial operon across 171 genomes.

The shades of gray represent the number of open reading frames (ORF) that are detected in each putative virulence gene.

Fig 9. Heatmap of the distribution of other virulence genes across 171 genomes.

The shades of gray represent the number of open reading frames (ORF) that are detected by in each putative virulence gene.