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

Anne-laure Moyne; Opeyemi U Lawal; Jeff Gauthier; Irena Kukavica-Ibrulj; Marianne Potvin; Lawrence Goodridge; Roger C Levesque; Linda J Harris

doi:10.1371/journal.pone.0291109

. 2023 Sep 7;18(9):e0291109. doi: 10.1371/journal.pone.0291109

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

Anne-laure Moyne ^1,², Opeyemi U Lawal ³, Jeff Gauthier ⁴, Irena Kukavica-Ibrulj ⁴, Marianne Potvin ⁴, Lawrence Goodridge ^3,⁵, Roger C Levesque ⁴, Linda J Harris ^1,^2,^*

Editor: Iddya Karunasagar⁶

PMCID: PMC10484465 PMID: 37676871

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.

Introduction

From 2001 to 2006, Salmonella enterica serovar Enteritidis was implicated in three outbreaks linked to raw almond consumption. Epidemiologic and traceback investigations of a 2000 to 2001 (denoted 2001) salmonellosis outbreak in Canada and in the United States identified a rare phage type (PT) of Salmonella Enteritidis, PT 30, from clinical samples, raw almonds sampled at retail, environmental (swab) samples at an almond processor and a huller-sheller facility, and environmental drag swabs obtained from multiple orchards in California [1]. Salmonella Enteritidis PT 30 was recovered in 2001 (three of six samples collected) [1]) and, in a subsequent study, at least two times in each year between 2002 and 2006 from environmental drag swabs collected in one of these orchards (eight to 96 samples collected each year) [2]. Raw almonds were epidemiologically linked to clinical cases of Salmonella Enteritidis PT 30 reported in Sweden in 2005 to 2006 (denoted 2006) [3] but Salmonella was not isolated from the implicated California almonds. Between 2003 and 2004 (denoted 2004), another rare phage type, Salmonella Enteritidis PT 9c, was linked to consumption of raw California almonds in the United States [4].

Excluding the farms linked to the 2001 outbreak, the prevalence and levels of Salmonella were determined in a multi-year survey of raw almond kernels. Samples of individual lots of raw almond kernels were collected as they were received by handlers (processors) located throughout the almond-growing regions of California from 2001 to 2007, and in 2010 and 2013 [5–7]. Inshell almonds were included in the survey in 2006 and 2007 [6]. The prevalence of Salmonella in 14,949 lots of raw almond kernels was 0.98% ± 0.29% over the 9 years (146 positive 100-g samples) [7]. Levels of Salmonella were estimated for 118 of the positive lots; mean and median levels of Salmonella were 1.14 ± 1.69 and 0.79 most probable number (MPN)/100 g, respectively, with single lots at 9.25 and 15.4 MPN/100 g [7]. Of the small number of raw inshell almond lots (455) evaluated in 2006 and 2007, 1.5% (seven 100-g subsamples) were positive [6]. Using classical serological nomenclature, the Salmonella isolates retrieved from these surveys were serotyped into 45 different serovars, including Salmonella Enteritidis PT 30 and PT 9c [5, 6].

Pulsed-field gel electrophoresis (PFGE), multilocus variable-number tandem repeat analysis (MLVA), and comparative genomic indexing (CGI) were applied to Salmonella Enteritidis strains associated with the 2001, 2004, and 2006 almond outbreaks, including clinical and environmental isolates [8]. The Salmonella Enteritidis PT 30 and PT 9c strains could be separated from each other and from other Salmonella Enteritidis phage types based on DNA enzyme restriction profiles, MLVA types, and genes identified by CGI. However, neither PFGE nor MVLA could discriminate among the Salmonella Enteritidis PT 30 isolates associated with the 2001 and 2006 almond-associated outbreaks. Salmonella Enteritidis PT 30 almond-associated outbreak strains could not be distinguished from epidemiologically unrelated Salmonella Enteritidis PT 30 clinical strains included in the study [8]. Among Salmonella Enteritidis PT 30 strains with identical genotypes, metabolic analyses with Biolog revealed differences between clinical and environmental isolates [8], indicating the discriminatory limit of the then current genotyping methods. Environmental Salmonella Enteritidis PT 30 isolates, collected between 2001 and 2006 from one of the 2001 outbreak-associated orchards, clustered in two groups based on the separation by PFGE of their XbaI-digested DNA [2].

Whole genome sequencing (WGS) has replaced PFGE for investigating foodborne outbreaks because of its higher resolution, and this methodology has been incorporated into routine public health surveillance since 2014 [9, 10]. Clonality of pathogen strains determined by WGS analyses can provide information about contamination during food production and distribution [11]. Different workflows have been developed to assess levels of genetic relatedness of foodborne pathogens by the National Center for Biotechnology Information (NCBI) with the Pathogen Detection Pipeline (https://www.ncbi.nlm.nih.gov/pathogens/), the U.S. Food and Drug Administration with the Center for Food Safety and Applied Nutrition CFSAN SNP Pipeline [12], and the U.S. Centers for Disease Control and Prevention with Lyve-SET [13], based on the specific needs of the respective agencies. The CFSAN SNP Pipeline creates high quality SNP matrices from WGS data that allow connection between clinical isolates to food or environmental isolates based on their evolutionary relationship [12]. Resident pathogen strains in facilities will have closely related WGS profiles, whereas transient pathogen strains will have unique or unrelated WGS profiles [11, 14].

The objective of the present study was a comparative genomic analysis of 171 Salmonella (45 serovars) isolated from raw inshell almonds and almond kernels in 9 years of surveys conducted between 2001 and 2013; 30 Salmonella Enteritidis PT 30 isolates recovered between 2001 and 2006 from a 2001 outbreak-associated almond orchard were included in the analysis. The CFSAN SNP Pipeline was selected to measure the genetic distances among the isolates. WGS was used to predict antimicrobial resistance (AMR), virulence genes, and presence of plasmid DNA.

Materials and methods

Isolate selection

Isolates were retrieved from enrichment of raw almond kernels and inshell almonds (survey isolates), and from swabs that were dragged across the floor of one of the 2001 outbreak-associated orchards (orchard isolates). A total of 15,505 ∼1-kg samples from different lots of raw almond kernels (14,949) and inshell almonds (455) were collected upon receipt at several almond processors located throughout California from the 2001–2005 [5], 2006–2007 [6], 2010 ([15]; present study), and 2013 ([7]; present study) harvests, as described previously. All samples were coded to keep the identities of the processors confidential; the geographic origins of the samples were unknown. The Safe Food Alliance, Kingsburg, CA (formerly American Council for Food Safety and Quality, Fresno, CA) analyzed a 100-g subsample from each lot of almonds by enriching for the presence of Salmonella [5]. In addition, the MPN of Salmonella was determined for 118 samples by enriching one or more additional samples weighing from ∼0.25 to 100 g. Salmonella isolates were stored at −80°C.

A total of 171 Salmonella survey isolates were selected for the present study: for each individual positive almond lot, a single Salmonella isolate retrieved from the initial 100-g subsample (153 positive samples, 148 isolates; three, one, and one isolate from 2001, 2002, and 2003, respectively, were lost), any isolates recovered from MPN or secondary enrichments that differed from the initial serotype (12), and unique isolates recovered from secondary enrichments of initially negative samples (11) (Tables 1 and S1 in S1 File). Traditional serotyping was done for each banked Salmonella by the California Animal Health and Food Safety Laboratory System (Davis, CA), and phage typing for Salmonella Enteritidis isolates was done by the National Veterinary Services Laboratory (Ames, IA) (Table 1). Salmonella Enteritidis PT 9c LJH1024 (obtained from Robert Mandrell, U.S. Department of Agriculture, Agricultural Research Service; RM4635 or G04-101 [8], raw almond isolate from the 2004 outbreak) and Salmonella Enteritidis PT 30 LJH0608 (raw almond isolate from the 2001 outbreak that was deposited to the American Type Culture Collection as ATCC BAA-1045) were also included in some of the analyses (S2 Table in S1 File).

Table 1. Results of the WGS serotyping and number of plasmids for Salmonella survey isolates retrieved from raw almonds.

Strain designation	Isolation year	Accession number	WGS serotyping		Traditional serotyping	Number of plasmids
Strain designation	Isolation year	Accession number	Subspecies	WGS serotyping	Traditional serotyping	Number of plasmids
LJH0651	2001	SRR23719048	enterica	Brandenburg	Brandenburg	0
LJH0652	2001	SRR23719047	enterica	Thompson	Thompson	0
LJH0653	2001	SRR23718942	enterica	Montevideo	Montevideo	4
LJH0654	2001	SRR23718931	enterica	Montevideo	Montevideo	0
LJH0655	2001	SRR23718920	enterica	Brandenburg	Brandenburg	0
LJH0656	2001	SRR23718909	enterica	Newport	Newport	2
LJH0657	2001	SRR23718898	enterica	Montevideo	Montevideo	0
LJH0658	2001	SRR23718887	enterica	Give	Nancy (Nchanga)	3
LJH0659	2001	SRR23719007	enterica	Montevideo	Montevideo	1
LJH0666	2002	SRR23718996	enterica	Typhimurium	Typhimurium	0
LJH0667	2002	SRR23719046	enterica	Senftenberg	Senftenberg	4
LJH0668	2002	SRR23718976	enterica	Give	Give	4
LJH0669	2002	SRR23718965	enterica	Typhimurium	Typhimurium	0
LJH0687	2002	SRR23718954	enterica	1,4,[5],12:i:-	1,4,[5],12:i:-	1
LJH0690	2002	SRR23719038	enterica	Oranienburg	Oranienburg	1
LJH0691	2002	SRR23719027	enterica	Oranienburg	Oranienburg	1
LJH0692	2002	SRR23719016	enterica	Worthington	Worthington	1
LJH0693	2002	SRR23718946	enterica	Heidelberg	Worthington	2
LJH0694	2002	SRR23718944	enterica	Oranienburg	Muenchen	1
LJH0695	2002	SRR23718943	enterica	Newport	Heidelberg	1
LJH0696	2002	SRR23718941	enterica	Thompson	Agona	0
LJH0697	2002	SRR23718940	enterica	Newport	Newport	1
LJH0698	2002	SRR23718939	enterica	Manhattan	Agona	1
LJH0713	2002	SRR23718938	enterica	Senftenberg	Senftenberg	0
LJH0714	2002	SRR23718937	enterica	Lomalinda	Lomalinda	1
LJH0715	2002	SRR23718936	enterica	Tennessee	Tennessee	1
LJH0716	2002	SRR23718935	enterica	Braenderup	Braenderup	2
LJH0717	2002	SRR23718934	enterica	Typhimurium	Typhimurium	0
LJH0718	2002	SRR23718933	enterica	Schwarzengrund	Schwarzengrund	3
LJH0719	2002	SRR23718932	enterica	Montevideo	Montevideo	1
LJH0720	2002	SRR23718930	enterica	Anatum	Anatum	1
LJH0721	2002	SRR23718929	enterica	Tennessee	Tennessee	0
LJH0722	2002	SRR23718928	enterica	Infantis	Infantis	0
LJH0724	2002	SRR23718927	enterica	Zerifin	Zerifin	4
LJH0725	2003	SRR23718926	enterica	Horsham	Brandenburg	0
LJH0726	2003	SRR23718925	enterica	Indiana	Indiana	7
LJH0738	2003	SRR23718924	enterica	1,4,[5],12:i:-	Typhimurium	1
LJH0739	2003	SRR23718923	enterica	Thompson	Thompson	0
LJH0740	2003	SRR23718922	enterica	Thompson	Thompson	1
LJH0741	2003	SRR23718921	enterica	Thompson	Thompson	1
LJH0742	2003	SRR23718919	enterica	Sandiego	Sandiego	1
LJH0751	2003	SRR23718918	enterica	Newport	Newport	1
LJH0752	2003	SRR23718917	enterica	Oranienburg	Othmarschen	1
LJH0753	2003	SRR23718916	enterica	Istanbul	Istanbul	3
LJH0754	2003	SRR23718915	enterica	Muenchen	Newport	1
LJH0759	2003	SRR23718914	enterica	Montevideo	Montevideo	0
LJH0760	2003	SRR23718913	enterica	Montevideo	Montevideo	1
LJH0761	2003	SRR23718912	enterica	Typhimurium	Typhimurium	1
LJH0762	2003	SRR23718911	enterica	Enteritidis	Enteritidis	1
LJH0783	2004	SRR23718910	enterica	Liverpool	Liverpool	0
LJH0784	2004	SRR23718908	enterica	Braenderup	Braenderup	1
LJH0787	2004	SRR23718907	enterica	Anatum	Anatum	3
LJH0788	2004	SRR23718906	enterica	Typhimurium	Typhimurium var. Copenhagen	1
LJH0789	2004	SRR23718905	enterica	Montevideo	Montevideo	0
LJH0790	2004	SRR23718904	enterica	Horsham	Horsham	0
LJH0791	2004	SRR23718903	enterica	Thompson	Thompson	1
LJH0792	2004	SRR23718902	enterica	Thompson	Thompson	1
LJH0793	2004	SRR23718901	enterica	Thompson	Thompson	1
LJH0794	2004	SRR23718900	enterica	Thompson	Thompson	1
LJH1011	2004	SRR23718899	enterica	Senftenberg	Senftenberg	0
LJH1012	2004	SRR23718897	enterica	Anatum	Anatum	2
LJH1013	2005	SRR23718896	enterica	Newport	Saintpaul	0
LJH1019	2005	SRR23718895	enterica	Give	Give	2
LJH1020	2005	SRR23718894	enterica	Montevideo	Montevideo	1
LJH1021	2005	SRR23718893	enterica	Heidelberg	Heidelberg	2
LJH1022	2005	SRR23718892	enterica	Mbandaka	Mbandaka	0
LJH1023	2005	SRR23718891	enterica	Enteritidis	Enteritidis	3
LJH1025	2005	SRR23718890	enterica	Typhimurium	Typhimurium	0
LJH1026	2005	SRR23718889	enterica	Manhattan	Untypeable	0
LJH1027	2005	SRR23718888	enterica	Muenchen	Muenchen	0
LJH1028	2005	SRR23718886	enterica	Enteritidis	Enteritidis	1
LJH1029	2005	SRR23718885	enterica	Enteritidis	Untypeable	1
LJH1030	2005	SRR23718884	enterica	Tennessee	Tennessee	0
LJH1043	2005	SRR23718883	enterica	Typhimurium	Typhimurium var. Copenhagen	1
LJH1044	2005	SRR23718882	enterica	Kentucky	Kentucky	2
LJH1045	2005	SRR23718881	enterica	Montevideo	Montevideo	0
LJH1046	2005	SRR23718880	enterica	Enteritidis	Enteritidis	1
LJH1047	2005	SRR23718879	enterica	Enteritidis	Enteritidis	0
LJH1048	2005	SRR23718878	enterica	Enteritidis	Enteritidis	1
LJH1049	2005	SRR23719008	enterica	Enteritidis	Enteritidis	1
LJH1052	2005	SRR23719006	enterica	Duisburg	Duisburg	3
LJH1054	2006	SRR23719005	enterica	Typhimurium	Typhimurium	1
LJH1055	2006	SRR23719004	enterica	Typhimurium	Typhimurium	1
LJH1056	2006	SRR23719003	enterica	Typhimurium	Typhimurium	1
LJH1058	2006	SRR23719002	enterica	Muenchen	Muenchen	0
LJH1059	2006	SRR23719001	enterica	Enteritidis	Enteritidis	1
LJH1063	2006	SRR23719000	enterica	Anatum	Anatum	1
LJH1067	2006	SRR23718999	diarizonae (IIIb)		III 50:k:-	0
LJH1068	2006	SRR23718998	enterica	Newport	Newport	0
LJH1070	2006	SRR23718997	enterica	Heidelberg	Heidelberg	2
LJH1071	2006	SRR23718995	enterica	Give	Give	2
LJH1076	2006	SRR23718994	enterica	Muenchen	Muenchen	0
LJH1080	2006	SRR23718993	enterica	Muenchen	Muenchen	0
LJH1082	2006	SRR23718992	enterica	1,4,[5],12:i:-	1,4,[5],12:i:-	1
LJH1083	2006	SRR23718991	enterica	Muenchen	Muenchen	0
LJH1084	2006	SRR23718990	enterica	Newport	Newport	1
LJH1085	2006	SRR23718989	enterica	Newport	Muenchen	0
LJH1087	2006	SRR23718988	enterica	Muenchen	Muenchen	0
LJH1088	2006	SRR23718987	enterica	Muenchen	Newport	0
LJH1089	2006	SRR23718985	enterica	Horsham	Horsham	0
LJH1090	2006	SRR23719045	enterica	Muenchen	Muenchen	0
LJH1094	2006	SRR23718986	enterica	Montevideo	Montevideo	0
LJH1095	2006	SRR23718984	enterica	1,4,[5],12:i:-	1,4,[5],12:i:-	1
LJH1096	2006	SRR23718983	enterica	Enteritidis	Enteritidis	2
LJH1097	2006	SRR23718982	enterica	Oranienburg	Oranienburg	1
LJH1098	2006	SRR23718981	enterica	1,4,[5],12:i:-	1,4,[5],12:i:-	1
LJH1099	2006	SRR23718980	enterica	Meleagridis	Meleagridis	0
LJH1100	2006	SRR23718979	enterica	Agona	Agona	1
LJH1101	2006	SRR23718978	enterica	Muenchen	Muenchen	0
LJH1102	2006	SRR23718977	enterica	Montevideo	Montevideo	0
LJH1103	2006	SRR23718975	enterica	Enteritidis	Enteritidis	1
LJH1104	2006	SRR23718974	enterica	Enteritidis	Enteritidis	1
LJH1105	2006	SRR23718973	enterica	Give	Give	1
LJH1106	2006	SRR23718972	enterica	Agona	Agona	2
LJH1107	2006	SRR23718971	enterica	Newport	Newport	2
LJH1108	2006	SRR23718970	enterica	Muenchen	Muenchen	0
LJH1109	2006	SRR23718969	enterica	Enteritidis	Enteritidis	4
LJH1133	2007	SRR23718968	enterica	Newport	Newport	0
LJH1134	2007	SRR23718967	enterica	Cerro	Cerro	0
LJH1135	2007	SRR23718966	enterica	Muenchen	Muenchen	0
LJH1136	2007	SRR23718964	enterica	Cerro	Cerro	0
LJH1137	2007	SRR23718963	enterica	Manhattan	Manhattan	1
LJH1138	2007	SRR23718962	enterica	Newport	Newport	2
LJH1139	2007	SRR23718961	enterica	Thompson	Thompson	0
LJH1140	2007	SRR23718960	enterica	Irumu	Irumu	0
LJH1141	2007	SRR23718959	enterica	Typhimurium	Typhimurium	2
LJH1142	2007	SRR23718958	arizonae (IIIa)		IIIa 18:z32:-	0
LJH1143	2007	SRR23718957	enterica	Oranienburg	Othmarschen	1
LJH1144	2007	SRR23718956	enterica	Typhimurium	I 4,12:i:-	3
LJH1145	2007	SRR23718955	enterica	Brandenburg	Brandenburg	2
LJH1146	2007	SRR23718953	enterica	Thompson	Thompson	0
LJH1147	2007	SRR23718952	enterica	Give	Bredeney	1
LJH1148	2007	SRR23718951	enterica	Newport	Newport	0
LJH1149	2007	SRR23718950	enterica	Cerro	Cerro	0
LJH1150	2007	SRR23719044	enterica	Senftenberg	Senftenberg	0
LJH1151	2007	SRR23719043	enterica	Muenchen	Untypeable	1
LJH1154	2007	SRR23719042	enterica	Senftenberg	Senftenberg	0
LJH1248-1	2010	SRR23719041	enterica	Newport	Newport	1
LJH1249-1	2010	SRR23719040	enterica	Give	Give	1
LJH1250-1	2010	SRR23719039	enterica	Infantis	Infantis	0
LJH1251-1	2010	SRR23719037	enterica	Give	Give	1
LJH1252-1	2010	SRR23719036	enterica	Infantis	Infantis	0
LJH1266-1	2010	SRR23719035	arizonae (IIIa)		II:17:g,t:-	0
LJH1267-1	2010	SRR23719034	enterica	Mbandaka	Mbandaka	0
LJH1268-1	2010	SRR23719033	enterica	Infantis	Infantis	0
LJH1269-1	2010	SRR23719032	enterica	Duisburg	Duisburg	1
LJH1270-1	2010	SRR23719031	enterica	Heidelberg	Heidelberg	2
LJH1271-1	2010	SRR23719030	enterica	Infantis	Infantis	0
LJH1272-1	2010	SRR23719029	enterica	Enteritidis	Enteritidis	1
LJH1273-1	2010	SRR23719028	enterica	Newport	Newport	1
LJH1276-1	2010	SRR23719026	enterica	Oranienburg	Othmarschen	1
LJH1277-1	2010	SRR23719025	enterica	Heidelberg	Heidelberg	2
LJH1278-1	2010	SRR23719024	enterica	Newport	Newport	1
LJH1618-1	2013	SRR23719023	enterica	1,4,[5],12:i:-	1,4,[5],12:i:-	1
LJH1619-1	2013	SRR23719022	enterica	Muenchen	I 6:8:d:z6	1
LJH1620-1	2013	SRR23719021	enterica	Muenchen	Untypeable	1
LJH1622-1	2013	SRR23719020	enterica	Heidelberg	Heidelberg	1
LJH1623-1	2013	SRR23719019	enterica	Montevideo	Montevideo	0
LJH1624-1	2013	SRR23719018	diarizonae (IIIb)	P:k:z35	Untypeable	0
LJH1628-1	2013	SRR23719017	enterica	Montevideo	Montevideo	1
LJH1629-1	2013	SRR23719015	enterica	Give	Give	1
LJH1630-1	2013	SRR23719014	enterica	Cerro	Cerro	2
LJH1631-1	2013	SRR23719013	enterica	Give	Give	2
LJH1633-1	2013	SRR23719012	enterica	Enteritidis	Enteritidis	2
LJH1660-1	2013	SRR23719011	arizonae (IIIa)		IIIa 41:z23: -	0
LJH1661-1	2013	SRR23719010	enterica	Muenchen	Muenchen	0
LJH1662-1	2013	SRR23719009	enterica	1,4,[5],12:i:-	Typhimurium	1
LJH1664-1	2013	SRR23718949	diarizonae (IIIb)		IIIb 50:r:z	0
LJH1665	2013	SRR23718948	enterica	1,4,[5],12:i:-	1,4,[5],12:i:-	1
LJH1673	2013	SRR23718947	enterica	Enteritidis	Enteritidis	2
LJH1676	2013	SRR23718945	enterica	1,4,[5],12:i:-	Typhimurium	1

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Several 2001 outbreak-associated almond orchards sampled by investigators during the 2001 outbreak investigation were positive for Salmonella Enteritidis PT 30 [1]. One of these orchards was sampled every year from 2001 [1] through 2006 [2]. Briefly, sterile gauze swabs attached to a string and soaked in full-strength evaporated skim milk were pulled along the orchard floor in a standardized manner. Four individual swabs were pooled and a procedure designed for recovering Salmonella from poultry houses was used to enrich the samples. Three of six (50%) pooled swab samples collected in 2001 and 53 of 228 (23%) samples collected between 2002 and 2006 were positive for Salmonella; every isolate was identified as Salmonella Enteritidis PT 30. A total of 30 Salmonella Enteritidis PT 30 orchard isolates from 2001 (3), 2002 (12), 2003 (10), 2005 (2), and 2006 (3) were analyzed in the present study (S2 Table in S1 File). One orchard isolate from 2002 and all orchard isolates from 2004 (25) were not available and thus not included.

Using the pathogen detection tool associated with the NCBI database (https://www.ncbi.nlm.nih.gov/pathogens), the sequence read archive (SRA) data were downloaded for 12 Salmonella Enteritidis PT 30 clinical isolates from the 2001 almond outbreak and for four clinical isolates from the 2006 almond outbreak (S3 Table in S1 File).

Whole genome sequencing

Isolates were retrieved from frozen glycerol stock and plated on tryptic soy agar (TSA). Following overnight incubation at 37°C, one colony was inoculated into 2 ml of tryptic soy broth (TSB) and incubated for 24 h at 37°C, with shaking at 120 rpm, before being pelleted by centrifugation at 14,000 × g for 2 min. Genomic DNA, for the isolates in S2 Table (S1 File), was extracted with the QIAamp DNA minikit (Qiagen, Valencia, CA) following the manufacturer’s directions. The 150-bp paired-end libraries were constructed for each purified DNA with the Illumina Nextera DNA flex library kit following the manufacturer’s directions (Illumina Inc., San Diego, CA). Pooled samples were sequenced on an Illumina 4000 HiSeq system by the DNA Technologies and Expression Analysis Core at the UC Davis Genome Center. DNA, for all the survey isolates, was sequenced as described by Emond-Rheault et al. in 2020 [16]. All sequence data obtained in this study were deposited to the NCBI pathogen database under the BioProject accession number PRJNA941918 (Tables 1 and S2 in S1 File) and PRJNA951760 (S2 Table in S1 File).

Quality control and genome assembly

Raw read quality was assessed with FastQC (v0.11.8) (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Illumina adapter sequences and low-quality sequences were trimmed using Trimmomatic version 0.36 [17]. Reads were assembled de novo with SPAdes version v3.13.0 Genome Assembler [18]. Draft Salmonella genome assemblies were serotyped with SeqSero [19] and by aligning with BLASTn against the nonredundant nucleotide sequence database.

Core genome SNP typing

De novo genomes were used to build core genome single nucleotide trees using the Parsnp aligner v1.2 with default parameters and the requirement for all genomes to be included in the analysis [20]. The 171 Salmonella genomes from the survey were mapped to the complete reference genome Salmonella Typhimurium str. LT2 (NCBI accession: AE006468), resulting in an alignment of 50% of the core genome. The whole-genome phylogeny was constructed with FastTree2 [21]. iTOL (http://itol.embl/de/) was used to visualize the tree and annotate it with the AMR genes [22].

Genetic distance

Genetic distance between multiple isolates of the same serovar was evaluated as the number of SNP differences detected with the CFSAN SNP Pipeline [12]. The CFSAN SNP Pipeline v2.0.2 was installed on a local ubuntu platform with all the executable software dependencies. Prior to analyzing our data, we used the data set provided for testing the reproducibility of the software to confirm that our installation of the CFSAN SNP Pipeline was correct. Reference-based alignments were created for a set of samples and used to generate the SNP matrix. Because the software was developed for closely related genome sequences, where available, complete assembled reference genomes were downloaded from NCBI (20; S4 Table in S1 File). The phylogenies were inferred with MEGA7 [23] using the neighbor-joining method [24] based on the obtained SNP matrix formatted as a FASTA file generated by the CFSAN SNP pipeline v2.0.2.

Virulence and antibiotic resistance (AMR) gene prediction

The presence of resistance genes, as well as point mutations, were determined using ResFinder 4.1 (Center for Genomic Epidemiology, https://cge.food.dtu.dk/services/ResFinder) with a setting threshold of 90% and minimum length of 60% [25–27]. The assembled draft genomes for the survey isolates (171) were used as an input to identify Salmonella AMR genes associated with resistance to aminoglycoside, β-lactam, chloramphenicol, colistin, fluoroquinolone, fosfomycin, glycopeptide, macrolide, sulfonamide, tetracycline, and trimethoprim antibiotics. The VF analyzer pipeline was used to screen the assembled draft genomes against the Virulence Factor Database (VFDB) for potential virulence factors [28].

Plasmid detection and reconstruction

Plasmids from genome assemblies were typed and reconstructed using MOB-suite v3.1.0 with the default parameters [29, 30]. To determine the AMR and virulence genes that were plasmid-borne, the reconstructed plasmids were screened against the CARD (https://card.mcmaster.ca) and VFDB [28] databases, respectively, using Abricate v0.5 (https://github.com/tseemann/abricate) with the same parameters as described above.

Results

Phylogenetic analysis of Salmonella isolates retrieved from raw almonds

Genomes were initially compared with Parsnp because the CFSAN SNP Pipeline is not recommended for relatively distant bacteria (greater than a few hundred SNP differences). A maximum-likelihood phylogeny tree was constructed with Parsnp based on alignment of the 171 Salmonella assembled genomes [20]. All the isolates belonged to the species enterica, with most (165) belonging to the subspecies enterica (Fig 1 and Table 1). Three isolates were classified as subspecies diarizonae and three as subspecies arizonae. The phylogenetic tree clustered the isolates by serovar (Fig 1). The serovars identified by classical serotyping matched the serovars predicted by WGS for 92% of the isolates (Table 1). However, 14 isolates clustered with serotypes that differed from those to which they were initially assigned by traditional serotyping (Table 1). To eliminate the possibility of manipulation errors, these isolates were resequenced and their serotype was confirmed with SeqSero and with BLASTn against the nonredundant nucleotide sequence database. Results were consistent with the initial WGS serovar prediction.

Fig 1 — 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.

Based on the core genome SNP typing, a total of 32 unique Salmonella serovars were identified. Of these, 22 Salmonella serovars were isolated two or more times between 2001 and 2013: Enteritidis (n = 16), Muenchen (n = 16), Newport (n = 15), Montevideo (n = 14), Typhimurium (n = 12), Thompson (n = 11), Give (n = 10), 1,4,[5],12:i- (n = 9), Oranienburg (n = 7), Heidelberg (n = 6), Infantis (n = 5), Senftenberg (n = 5), Anatum (n = 4), Cerro (n = 4), Brandenburg (n = 3), Duisburg (n = 3), Horsham (n = 3), Manhattan (n = 3), Tennessee (n = 3), Agona (n = 2), Braenderup (n = 2), and Mbandaka (n = 2) (Fig 1 and Table 1). Three isolates initially identified as Salmonella Othmarschen (LJH0752, LJH1143, and LJH1276-1) clustered with Salmonella Oranienburg. Both serotypes have a similar antigenic formula, 6,7,14:m,t:-, which makes them difficult to distinguish by serological methods [31]. Serovar 1,4,[5],12:i:- is a monophasic variant of Salmonella Typhimurium, and three of 14 isolates initially identified as Typhimurium (LJH0738, LJH1662-1, LJH1676) clustered with 1,4,[5],12:i:-. One Salmonella initially identified as 1,4,[5],12:i:- (LJH1144) clustered with Typhimurium.

Among the five isolates that were untypeable by classical serotyping, two clustered with serovar Muenchen (LJH1151, LJH1620-1), one with Enteritidis (LJH1029), and one with Manhattan (LJH1026). A single untypeable isolate (LJH1624-1) clustered with Salmonella diarizonae (LJH1664) and was identified as Salmonella diarizonae with BLASTn and SeqSero [19]. Fifteen unique serotypes, predicted with BLASTn and SeqSero, matched the serological serotyping. It was difficult to predict some serovars due to the limited number of representative genomes in the SeqSero database. Salmonella Zerifin LJH0724 was identified as Salmonella Istanbul with SeqSero and clustered with the Istanbul isolates in the Parsnp phylogeny tree (Fig 1). Conflicting serotyping results between traditional methods and WGS have been reported in previous studies [32–35]. Serotyping based on WGS has been increasingly used by public health laboratories and federal agencies to replace the current standard of phenotypic serotyping [10, 32, 34, 36]. In the present study, serotype was assigned based on the WGS analysis when the serovar assignment was identical among the phylogenetic relationship, SeqSero, and BLASTn analyses. Except for a single strain of Salmonella Zerifin, all isolates (170 of 171) were assigned a serotype based on the WGS analysis.

Genetic distance within each serovar

The CFSAN SNP Pipeline was used to evaluate the genetic distance among multiple isolates within each serotype. Salmonella Enteritidis isolates clustered in three groups by phage type: PT 8, PT 9c, and PT 30 (Fig 2). All five Salmonella Enteritidis PT 8 isolates were retrieved in 2005 from separate almond lots and had less than three SNP differences, classifying them as clonal isolates. Salmonella Enteritidis PT 9c isolated in 2005 (LJH1028) and 2010 (LJH1272), differed by 5 and 13 SNPs, respectively, from a 2004 Salmonella Enteritidis PT 9c outbreak isolate (LJH1024; Fig 2).

The genomes of Salmonella Enteritidis PT 30 recovered from survey almonds (eight isolates (Table 1): LJH0762 [2003], LJH1023 [2005], LJH1104 [2006], LJH1059 [2006], LJH1096 [2006], LJH1109 [2006], LJH1633 [2013], LJH1673 [2013]), the 2001 outbreak-associated orchard (30 isolates; S2 Table in S1 File), and a 2001 outbreak-associated almond isolate (LJH0608) were compared to Salmonella Enteritidis PT 30 genomes of clinical isolates from almond outbreaks in 2001 (12 isolates) and 2006 (four isolates) (S3 Table in S1 File).

Salmonella Enteritidis PT 30 isolates formed two clusters (Fig 2). One consisted of a single survey isolate (LJH0762), recovered in 2003, that differed from LJH0608 by 48 SNPs (Fig 2 and S5 Table in S1 File). All other survey and clinical isolates (n = 38) clustered in a single group with LJH0608 that differed from each other by ≤18 SNPs (Fig 2) indicating that the isolates are from a common origin. Almond isolates from 2001 to 2013 had 2 to 13 SNP differences compared with the 2001 outbreak-associated almond isolate Salmonella Enteritidis PT 30 LJH0608 (Fig 2 and S5 Table in S1 File). Although this isolate was recovered from recalled almonds in 2001, the almonds were harvested in the fall of 2000 [1], a span of 14 years (2000–2013). The orchard isolates from 2001 to 2006 differed by 0 to 12 SNPs within their genomes and by 3 to 13 SNPs with the clinical genomes. The SNP differences ranged from zero to eight within the 12 clinical isolates from the 2001 outbreak and from one to 13 within the four clinical isolates from the 2006 outbreak. Among the clinical isolates from 2001 and 2006, the SNP differences ranged from four to 13, indicating that the isolates are from a common origin.

Almond, orchard, and clinical isolates of Salmonella Enteritidis PT 30 isolated from 2001 through 2013 are closely related strains. The persistence of Salmonella Enteritidis PT 30 in an almond orchard over 6 years was reported previously [2]. The SNP analysis confirmed the PFGE results obtained for these isolates. Almonds from the 2001 outbreak-associated orchards were purposefully excluded from the raw almond survey. Because survey samples were coded and the sources unknown, it is possible that samples harvested from outbreak-associated orchards were inadvertently included. It is also possible that survey almonds were cross contaminated with almonds harvested from outbreak-associated orchards via harvest equipment or at a common almond huller-sheller, or that Salmonella Enteritidis PT 30 was spread over a broader geographic region than recognized as associated with the 2001 outbreak.

Salmonella Enteritidis was not the only serovar for which clonal isolates were recovered from almonds in different years. Salmonella Montevideo survey isolates clustered into three groups, with more than 100 SNPs between them (Fig 3). Within each of these clusters there were isolates separated by one or more years (including isolates from 2001 and 2013) that differed by ≤9 SNPs, indicating that they share a common ancestor. Salmonella Newport (S6 Table in S1 File) and Salmonella Muenchen (S7 Table in S1 File) each clustered into two groups separated by more than 100 SNPs. A small number of SNP differences (<3) between Salmonella Newport genomes (S6 Table in S1 File) were identified in isolates retrieved in 2003 (LJH0751), 2006 (LJH1084), 2007 (LJH1133), 2010 (LJH1248, LJH1273 and LJH1278). Nine isolates of Salmonella Muenchen were retrieved in 2006; eight of these isolates from six different almond lots had nearly identical genomes, with ≤3 SNP differences (S7 Table in S1 File), and differed from single isolates in 2007 (LJH1135) and 2013 (LJH1661) by ≤8 SNPs. Closely related genomes for single isolates of Salmonella Anatum (S8 Table in S1 File) and Salmonella Thompson (S9 Table in S1 File) were identified 2 and 1 years apart, respectively. Several Salmonella Oranienburg (S10 Table in S1 File) and Salmonella Typhimurium (Fig 4) were identified 5 and 3 years apart, respectively. Closely related genomes (≤13 SNPs) were identified for isolates retrieved from separate almond lots during the same year for Salmonella serovar Cerro (S11 Table in S1 File), Give (S12 Table in S1 File), Infantis (S13 Table in S1 File), Heidelberg (S14 Table in S1 File) and Tennessee (S15 Table in S1 File). For Salmonella serovar Brandenburg (S16 Table in S1 File), Braenderup, Manhattan (S17 Table in S1 File), Mbandaka, and Senftenberg (S18 Table in S1 File), isolates were separated by more than 13 SNPs.

Fig 3 — The branch lengths represent the SNP distances among the isolates.

Fig 4 — The branch lengths are representative of the SNP distances among the isolates.

Multiple SNP-based approaches have been developed to analyze the large number of short reads produced by various sequencing platforms [37, 38]. The CFSAN Pipeline was selected because it is less sensitive to coverage changes [37] and has good discriminative power. Its high resolution, however, strongly depends on an appropriate reference genome. In addition to outbreak source attribution, this tool can reveal similarity among isolates and their persistence in food facilities or in the production environment [14, 39].

Differences of more than 100 SNPs were sometimes observed among isolates within each serovar. Survey isolates that were genetically similar (≤13 SNPs) were recovered in multiple years, up to 13 years for Salmonella Enteritidis PT 30 and Salmonella Montevideo and 10 years for Salmonella Newport. Because the survey samples were collected after hulling and shelling and prior to entering a processing facility or storage, the contamination source would have to be at production or harvest (orchard), during postharvest handling (transportation to huller, during storage, or during hulling and shelling), or transportation from huller to processor. Although the exact geographic locations of the almond survey samples were unknown, clonal strains of Salmonella Enteritidis PT 8 (five; 2005) and Salmonella Muenchen (eight; 2006) were isolated from different lots of almonds in the same year.

The diversity of serovars and unique strains within serovars was expected in a survey that likely reflects broad environmental contamination (e.g., almond orchard during production or harvest). Common harvest and postharvest practices may also lead to distribution of Salmonella in almonds from geographically diverse orchards that share the same equipment or facilities. At maturity, almonds are shaken to the ground where they dry for several days. They are then harvested by windrowing and sweeping off the orchard floor. The in-hull, inshell almonds are then transported to facilities where the hull and shell are removed. Kernels mix with hulls and shells before sorting, separation, and bulk transportation to processing facilities. Once kernels are delivered to almond processing facilities, commingling of almond lots may occur prior to or during storage. These practices may explain the clusters of Salmonella Enteritidis PT 8 and Salmonella Muenchen isolated from different lots in 2005 and 2006.

U.S. regulations were implemented in 2007 that require all California-grown almonds sold in North America (U.S., Canada, and Mexico) to be processed with a treatment capable of achieving a minimum 4-log reduction in Salmonella [40]. While there have been outbreaks associated with almond-containing products such as blended nut butters, none have been associated with contaminated almonds since 2006, likely due to effective implementation of these regulations [7, 15].

The persistence of Salmonella has been described for other pre- and postharvest scenarios [11, 41, 42]. A narrow range of Salmonella serovars has been associated with California pistachio outbreaks, outbreak investigations, and industry and retail surveys [41, 43]. Pistachio-associated isolates of Salmonella Senftenberg and Salmonella Montevideo recovered over multiple years (2009 to 2017) and from multiple facilities differed (within each serovar) by 0 to 31 SNPs, and the authors suggested that the organisms may have established residence in the primary production environment or orchards [41].

Plasmid prediction and characterization

Plasmids contribute significantly to the emergence and spread of genes encoding AMR, virulence, and other metabolic functions in multiple scales across Salmonella serotypes [16, 44]. Plasmid carriage in the Salmonella strains under study were assessed by screening and reconstructing the plasmid sequences from assembled genomes using the clustered plasmid reference database-based pipeline [29, 30]. A total of 106 of the 171 Salmonella isolates (62%) carried one to seven plasmids (total plasmids 161; Table 1) with sizes of 1,030 bp to 303,322 bp (Fig 5A and S19 Table in S1 File). Using the presence of relaxase and mate-pair formation marker genes and/or oriT sequence, most of these plasmids were predicted to be either mobilizable (n = 51/161; 32%) or conjugative (n = 83/161; 52%) (Fig 5A). In total, 61 plasmid clusters with 27 different plasmid families were identified, with IncFII and IncFIB being the most predominant types in the collection (Fig 5B).

Fig 5 — (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.

Thirty eight Salmonella strains distributed across six serovars (Duisburg, Enteritidis, Lomalinda, Muenchen, Typhimurium, and 1,4,[5],12:i:-) carried plasmids that contained two to seven virulence genes, but no AMR genes (S20 Table in S1 File). The IncFIB-IncFII plasmid family combination was common among these strains (Fig 6), predominantly in Salmonella Enteritidis (n = 15), Typhimurium (n = 8), and 1,4,[5],12:i:- (n = 9). Comparative analysis of the plasmid sequences with known plasmids using mash [45] and BLASTn revealed that these plasmids are similar (nucleotide sequence homology = 100%) to plasmid pCFSAN076214_2 (accession number CP033342.1) described previously in Salmonella Enteritidis strain ATCC BAA-1045 isolated from raw almonds (also LJH608 in this study) [46] and to plasmid p11-0972.1 (accession number CP039855.1) reported in S. enterica serovar 1,4,[5],12:i:- recovered from a human stool sample [47]. IncF plasmids are among the most common plasmids found in Salmonella and are reported to carry multiple antibiotic resistance and/or virulence genes, suggesting their role in the dissemination of these genes across Salmonella serotypes and Enterobacteriaceae by extension [48, 49].

Thirteen Salmonella strains belonging to seven serotypes (Agona, Anatum, Heidelberg, Istanbul, Newport, Typhimurium, and Zerifin) carried plasmids with at least one AMR gene but no virulence genes. The most common among these was within the IncC plasmid family and was associated with six to 10 AMR genes (S20 Table in S1 File). The IncC plasmid identified in the present study was predicted to be conjugative, predominant in Salmonella Anatum and Salmonella Newport (Fig 6) and was almost indistinguishable (99.99% by BLASTn) from Salmonella Anatum plasmid pSAN1-1736 (accession number: CP014658.1) [50]. IncC plasmids are known to be widely distributed across Salmonella serotypes from diverse sources and often carry multiple AMR genes [49].

A unique Salmonella Kentucky strain LJH1044 carried a large conjugative plasmid (146 Kb) with IncFIB-IncFIC-rep_cluster_2244 plasmid replicons and contained multiple virulence and AMR genes (Fig 6 and S20 Table in S1 File). This plasmid carried three AMR genes encoding resistance to aminoglycoside and tetracycline, a complete iroBCDEN operon that encodes salmochelin siderophore [51], and an iucABCD-iutA operon that has been described to be associated with aerobactin synthesis essential for virulence and stress response in Salmonella [52] (S20 Table in S1 File). Comparative analysis showed that this plasmid had 99.99% nucleotide sequence similarity to plasmid pCVM29188_146 (accession number CP001122.1) reported previously in several Salmonella Kentucky strains from poultry in the United States [53, 54].

Antimicrobial resistance profile

Among the isolates retrieved from the almond survey, a total of 24 AMR genes were identified with the ResFinder and CARD database, which classified into nine different antimicrobial protein groups: aminoglycosides, β-lactams, colistin, fosfomycin, glycopeptide, phenicol, sulfonamide, tetracycline, and trimethoprim. One gene, aac(6’)-Iaa, which confers resistance to aminoglycosides, was detected in the chromosome of all Salmonella subspecies diarizonae (3) and enterica (165) but not in arizonae (3) (Fig 1). The frequency of Salmonella enterica subspecies enterica isolates that carried an aminoglycoside acetyltransferase aac(6’)-Iaa has been reported to be high (>95%) in multiple WGS analysis [55–57]. However, in a surveillance study of non-typhoidal Salmonella enterica, 11 isolates out of 3,491 (0.3%) showed phenotypic resistance to an aminoglycoside antimicrobial [55].

The other 23 AMR genes were detected in 35 isolates, with fosA7 being the most common (Fig 1). The gene fosA7, which confers resistance to fosfomycin, a broad-spectrum cell wall synthesis inhibitor, was first identified in the chromosome of Salmonella Heidelberg isolated from broiler chickens [58]. In the present study, all Salmonella Heidelberg isolates (n = 6), and some isolates of Salmonella serovars Agona (n = 2), Meleagris (n = 1), Montevideo (n = 8), Oranienburg (n = 2), and Tennessee (n = 3), carried the chromosomal fosA7 gene. Ten aminoglycoside resistance genes were detected in 16 isolates: aac(6’)-Iaa, aadA2, aadA3, aadA7, aadA12, aadA13, ant(2”)-Ia, aph(3")-IIa, aph(3’’)-Ib, aph(6)-lc, and aph(6)-Id. The bla_CMY-2 and bla_CARB-2 genes, which confer resistance to β-lactams, and the floR gene, which confers resistance to phenicol, were found in nine isolates. Three tetracycline efflux resistance genes were identified in 16 isolates: tetA, tetB, and tetG. Sulfisoxazole resistance, encoded by sul1 or sul2, was detected in 11 isolates. The dihydrofolate reductase resistance gene, dfrA12, which confers resistance to trimethoprim, was detected in one Salmonella Newport isolate. The plasmid-mediated colistin resistance and phosphoethanolamine transferase mcr-9.1 gene was detected in one Salmonella Agona isolate. Resistance to bleomycin, encoded by the bleomycin-binding protein gene (ble), was predicted for one Salmonella Heidelberg isolate. All the isolates, except Salmonella serovars Enteritidis, Irumu, Lomalinda, Typhimurium, and 1,4,[5],12:i-, contained a missense mutation in parC associated with resistance to quinolone. Multiple mutations in the quinolone resistance determining region are usually required to confer resistance to ciprofloxacin, but one mutation confers resistance to nalidixic acid [59]. One Salmonella Senftenberg isolate had a point mutation in the 16S rRNA that is thought to confer resistance to spectinomycin.

Most of the predicted AMR genes were identified in a small number (16 out of 171; 9%) of survey isolates and were plasmid encoded in 11 of 16 cases (Fig 1). Multidrug-resistant isolates (putative resistance to at least one antibiotic in three or more drug classes; https://www.cdc.gov/narms/resources/glossary.html) were identified among Salmonella serovars Agona (LJH1100 and LJH1106), Anatum (LJH0720, LJH0787, LJH1012, and LJH1063), Heidelberg (LJH1622), Newport (LJH0656, LJH1138, and LJH1107), and Typhimurium (LJH0788 and LJH1141) (Fig 1). Antibiotic resistance profiles by the calibrated dichotomous sensitivity method were determined for Salmonella isolated from 2001 through 2005 but not for isolates from 2006, 2007, 2010, and 2013. Ten of the Salmonella survey isolates from 2001 to 2005 were resistant to three or more antibiotics [5]. Resistance genotype and phenotype correlated highly for five of these isolates: Salmonella Anatum (n = 3), Salmonella Istanbul (n = 1), and Salmonella Typhimurium var. Copenhagen (n = 1).

Analysis of virulence factors

Salmonella serovars infect a wide range of hosts with different degrees of disease severity, with Enteritidis, Newport, Typhimurium, Javiana, and 1,4,[5],12:i- being significantly more likely to cause illness in humans in the United States [60]. Differences in virulence factors contribute to the severity and outcome of salmonellosis and can be specific to serovars [60]. The Virulence Factor Database (VFDB) was used to detect a total of 303 virulence genes among the 171 Salmonella assembled genomes (Figs 7–9). Genes were classified under major virulence factors, including the secretion system, fimbrial and non fimbrial adherence, macrophage inducible genes, magnesium uptake, serum resistance, stress proteins, toxins, immune invasion, and two component regulatory systems. The Salmonella pathogenicity island 1 (SPI-1) and 2 (SPI-2), responsible for the type III secretion system, are ubiquitous in S. enterica subsp. enterica [60] and were common to all 171 survey isolates (Fig 7).

Fig 7 — The shades of gray represent the number of open reading frames (ORF) that are detected in each putative virulence gene.

Fig 9 — The shades of gray represent the number of open reading frames (ORF) that are detected by in each putative virulence gene.

Among the fimbrial adherence factors, the genes encoding the curli fimbriae, csgA, csgB, and csgE, the bcfABCDEFG operon, and the genes that encode type 1 fimbriae, fimA, fimB, fimC, fimD, fimH, fimI, fimW, and fimZ, were also present in all the isolates (Fig 8). The two- component regulatory system phoP-PhoQ genes and the magnesium uptake genes, mgtB-mgtC, (part of SPI-3) were present in all the isolates (Fig 9). All the isolates had the microphage inducible gene, mig-14, but mig-5 was mainly detected in isolates of Salmonella serovars Enteritidis, Typhimurium, and 1,4,[5]12:I:- (Fig 9).

Fig 8 — The shades of gray represent the number of open reading frames (ORF) that are detected in each putative virulence gene.

The typhoid toxin genes, cdtB and pltA, originally identified in serotype Typhi, were found in all Salmonella serovar Brandenburg, Duisburg, Horsham, Montevideo, Sandiego, and Schwartzengrund isolates and in nine of 10 Salmonella Oranienburg isolates (Fig 9). However, pltB, required for forming holotoxin, was not present in any of these isolates. In Salmonella Horsham isolates, two genes were identified as homologs of the enterotoxin hemolysin genes of Escherichia coli (hylE/clyA) (Fig 9).

A virulence plasmid that harbored a combination of pef, rck, and spv virulence genes was identified in 23% of the isolates (S20 Table in S1 File). The assembly of the major Pef fimbriae depends on the pefBACDorf5orf6 operon, which encodes PefA fimbriae subunit, PefC usher protein, and the pefD periplasmic chaperone [61]. Eight of 10 Salmonella Typhimurium and all nine Salmonella 1,4,[5]12:I:- isolates carried a plasmid encoding pefA, pefB, pefC, and pefD genes (Fig 8 and S20 Table in S1 File).

The spv genes play a role in suppression of the innate immune response and are often associated with invasive disease and increased virulence [62, 63]. The spv genes were detected in all Salmonella serovar Lomalinda, Enteritidis, and 1,4,[5]12:I:- isolates, in eight of 10 Salmonella Typhimurium isolates, and one of 10 Salmonella Give isolates (Fig 9 and S20 Table in S1 File). The rck gene, which provides protection against the complement-mediated immune response of the host [64], was found in one of 10 Salmonella Give isolates, in all Salmonella Enteritidis PT 30 and PT 8 isolates, in eight of 10 Salmonella Typhimurium isolates, and all Salmonella 1,4,[5]12:I;- isolates (Fig 9).

This study provides one of the first in-depth longitudinal characterizations of Salmonella strains isolated from a single product (almonds) or production environment (almond orchard), in a single geographical region (Central California). This isolate collection is important for understanding Salmonella populations in a significant food production region of the United States. Several clonal strains of Salmonella were isolated over multiple years, adding to a growing body of evidence that enteric pathogens may persist over long periods of time (years) in agricultural environments and in postharvest or food processing facilities (https://www.cdc.gov/ncezid/dfwed/outbreak-response/rep-strains.html) [41, 42]).

Supporting information

S1 File

(XLSX)

Click here for additional data file.^{(123.2KB, xlsx)}

Acknowledgments

We thank Sylvia Yada for editing the manuscript and Vanessa Lieberman for technical assistance.

Data Availability

All the data are in the NCBI database with the following links: https://www.ncbi.nlm.nih.gov/bioproject/PRJNA941918 https://www.ncbi.nlm.nih.gov/bioproject/PRJNA951760

Funding Statement

LJH 18-HarrisL-AQFSS-01 Almond Board of California (https://www.almonds.com) LG/RCL Genome Canada (https://genomecanada.ca) and Genome Quebec (https://www.genomequebec.com/en/home/) The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS One. doi: 10.1371/journal.pone.0291109.r001

Decision Letter 0

Iddya Karunasagar

27 Jun 2023

PONE-D-23-14605Genetic diversity of Salmonella enterica isolated from raw California almonds and from an almond orchard over 13 YearsPLOS ONE

Dear Dr. Harris,

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Reviewer #1: PONE-D-23-14605

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

This study compared the whole genome sequences of 171 Salmonella isolates that included 30 isolates of S. Enteritidis PT30 involved in almond-associated outbreaks. SNP analysis, serovar identification, antimicrobial resistance genes, plasmid types and virulence genes were determined. The main goal of this study was to see if S. Enteritidis PT30 outbreak isolates could be distinguished from similar environmental isolates.

The study emphasizes the discriminatory power of WGS over traditional genotyping methods. The study identified clonal isolates of Salmonella Enteritidis and Salmonella Montevideo. The persistence of clonal strains of certain serovars suggest contamination of post-harvest processing areas with these strains.

1. The manuscript is straightforward presentation of the WGS comparison. The study assumes significance in few contexts; that Salmonella Enteritidis PT 30 isolates belong to a long time line of over 13 years, comprise of outbreak strains and their environmental counterparts, and as the result suggest, these strains were phylogenetically closely related.

The study aimed to solve three important issues with the traditional genotyping methods as stated in the introduction section and listed below.

2. Lines 80-82:. “However, neither PFGE nor MVLA could discriminate among the Salmonella Enteritidis PT 30 isolates associated with the 2001 and 2006 almond-associated outbreaks”

Now, the WGS analysis clustered (lines 275-276) clustered them together in one group with ≤18 SNPs. Is this not similar to PFGE results? Or does a difference of 18 SNPs make them different from each other?

3. Lines 82-84: Salmonella Enteritidis PT 30 almond associated outbreak strains could not be distinguished from epidemiologically unrelated Salmonella Enteritidis PT 30 clinical strains included in the study

Again, the fact that the WGS analysis put them together in one group suggests the same thing.

4. Lines 87-89: Environmental Salmonella Enteritidis PT 30 isolates, collected between 2001 and 2006 from one of the 2001 outbreak-associated orchards, clustered in two groups based on the separation by PFGE of their XbaI-digested DNA

As could be seen from the results in lines 274-276, one survey isolate formed a separate group.

5. Lines 294-295: With less than ≤18 SNPs, are they still termed “closely related strains”. In a �4 Mb genome sequence, what would be the NGS contribution to SNPs? Although this depends on the depth of sequencing, still there would be contribution from the sequencing itself.

6. Although AMR genes were identified from the WGS, these are not correlated with the antibiotic resistance phenotype of isolates.

7. Only two pathogenicity islands (SPI-1 & SPI-2) were identified in WGS. What about the rest?

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Reviewer #1: No

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PLoS One. 2023 Sep 7;18(9):e0291109. doi: 10.1371/journal.pone.0291109.r002

Author response to Decision Letter 0

4 Aug 2023

PONE-D-23-14605

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

Editor and Reviewer comments are in italics. Author responses are not in italics.

Please include the following items when submitting your revised manuscript:

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Response: Done.

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Response: Done.

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JR 2. Thank you for stating the following in the Acknowledgments Section of your manuscript: "This research was supported by the Almond Board of California Grant 18-HarrisL536 AQFSS-01. The DNA Technologies and Expression Analysis Core at UC Davis Genome Center is supported by NIH shared Instrumentation Grant 1S10OD010786-01. L. Goodridge and RC Levesque were funded by Genome Canada and Genome Québec. We thank Sylvia Yada for editing the manuscript and Vanessa Lieberman for technical assistance."

We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows: "LJH 18-HarrisL-AQFSS-01 Almond Board of California (https://www.almonds.com) LG/RCL Genome Canada (https://genomecanada.ca) and Genome Quebec (https://www.genomequebec.com/en/home/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."

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Response: We have deleted the funding information from the Acknowledgement section in the manuscript.

Amended Funding Statement: “LJH 18-HarrisL-AQFSS-01 Almond Board of California (https://www.almonds.com), LG/RCL Genome Canada (https://genomecanada.ca) and Genome Quebec (https://www.genomequebec.com/en/home/), NIH shared Instrumentation Grant 1S10OD010786 (https://orip.nih.gov/construction-and-instruments/s10-instrumentation-programs). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."

JR 3. We note that you have stated that you will provide repository information for your data at acceptance. Should your manuscript be accepted for publication, we will hold it until you provide the relevant accession numbers or DOIs necessary to access your data. If you wish to make changes to your Data Availability statement, please describe these changes in your cover letter and we will update your Data Availability statement to reflect the information you provide.

Response: We included all relevant accession numbers in the manuscript and have now released those data.

JR 4. Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Response: Reference list has been reviewed and is complete and correct.

Reviewer #1:

The manuscript is straightforward presentation of the WGS comparison. The study assumes significance in few contexts; that Salmonella Enteritidis PT 30 isolates belong to a long timeline of over 13 years, comprise of outbreak strains and their environmental counterparts, and as the result suggest, these strains were phylogenetically closely related.

The study aimed to solve three important issues with the traditional genotyping methods as stated in the introduction section and listed below.

Response: Thank you for your comments and manuscript overview. As stated in the last paragraph in the introduction, the objective of the study was a comparative genomic analysis of almond-related Salmonella isolates. The study did not aim to solve issues with traditional genotyping methods. However, we have addressed each comment related to statements in the introduction below.

R1c1a Lines 80-82: “However, neither PFGE nor MVLA could discriminate among the Salmonella Enteritidis PT 30 isolates associated with the 2001 and 2006 almond-associated outbreaks”. Now, the WGS analysis clustered (lines 275-276) clustered them together in one group with ≤18 SNPs. Is this not similar to PFGE results? Or does a difference of 18 SNPs make them different from each other?

R1c1b Lines 82-84: Salmonella Enteritidis PT 30 almond associated outbreak strains could not be distinguished from epidemiologically unrelated Salmonella Enteritidis PT 30 clinical strains included in the study. Again, the fact that the WGS analysis put them together in one group suggests the same thing.

R1c1c Lines 87-89: Environmental Salmonella Enteritidis PT 30 isolates, collected between 2001 and 2006 from one of the 2001 outbreak-associated orchards, clustered in two groups based on the separation by PFGE of their XbaI-digested DNA. As could be seen from the results in lines 274-276, one survey isolate formed a separate group.

Response: The statement regarding the PFGE or MVLA was specific to clinical isolates of Salmonella Enteritidis PT 30 which could not be distinguished from each other using PFGE or MVLA (refers to Parker et al. [8]).

With one exception (LJH0762), we were also unable to distinguish the Salmonella Enteritidis PT 30 isolates assessed in the present paper by WGS (beyond 18 SNP differences). Our analysis consisted of 55 isolates: eight almond survey isolates, 30 orchard isolates and 16 outbreak isolates. We did not include some of the non-almond associated clinical isolates that were evaluated in the Parker et al [8] study and thus are unable to know if WGS could distinguish among all the isolates used in that study.

To clarify, we made minor modification to the concluding paragraph of the introduction to specify that 171 isolates (45 serovars) were evaluated (not just Salmonella Enteritidis PT 30). We provided additional information under “Genetic Distance Within Each Serovar” (copied below) to highlight the number of Salmonella Enteritidis PT 30 isolates that were assessed. We added a new supplemental table (S5 Table) that complements Figure 2 and provides more granularity to SNP differences. We also further clarified that almond isolates from 2001 to 2013 differed from the 2001 outbreak-associated almond isolate LJH0608 (from almonds harvested in 2000) by 2 to 13 SNPs and that this represents isolates collected over a span of 14 years.

We also modified the following sentence to remove the statement “with higher resolution”:

“The persistence of Salmonella Enteritidis PT 30 in an almond orchard over 6 years was reported previously [2]. The SNP analysis confirmed the PFGE results obtained for these isolates.”

The reviewer’s statement (R1c1c) regarding environmental Salmonella isolates was related to orchard isolates (refers to Uesugi et al., 2006 [2]). The comment refers to only the environmental samples retrieved from the outbreak-associated almond orchard. The orchard isolates from 2001 to 2006 differed by 0 to 12 SNPs in our analyses, which is now more clearly stated in the modified results section (copied below). We believe that the changes outlined above adequately address the reviewer’s comments.

“The genomes of Salmonella Enteritidis PT 30 recovered from survey almonds (eight isolates (Table 1): LJH0762 [2003], LJH1023 [2005], LJH1104 [2006], LJH1059 [2006], LJH1096 [2006], LJH1109 [2006], LJH1633 [2013], LJH1673 [2013]), the 2001 outbreak-associated orchard (30 isolates; S2 Table), and a 2001 outbreak-associated almond isolate (LJH0608) were compared to Salmonella Enteritidis PT 30 genomes of clinical isolates from almond outbreaks in 2001 (12 isolates) and 2006 (four isolates) (S3 Table).

Salmonella Enteritidis PT30 isolates formed two clusters (Fig 2). One consisted of a single survey isolate (LJH0762), recovered in 2003, that differed from LJH0608 by 48 SNPs (Fig 2, S5 Table). All other survey and clinical isolates (n = 38) clustered in a single group with LJH0608 that differed from each other by ≤18 SNPs (Fig 2) indicating that the isolates are from a common origin. Almond isolates from 2001 to 2013 had two to 13 SNP differences compared with the 2001 outbreak-associated almond isolate Salmonella Enteritidis PT 30 LJH0608 (Fig 2, S5 Table). Although this isolate was recovered from recalled almonds in 2001, the almonds were harvested in the fall of 2000 [1], a span of 14 years (2000–2013). The orchard isolates from 2001 to 2006 differed by 0 to 12 SNPs within their genomes and by 3 to 13 SNPs with the clinical genomes. The SNP differences ranged from zero to eight within the 12 clinical isolates from the 2001 outbreak and from one to 13 within the four clinical isolates from the 2006 outbreak. Among the clinical isolates from 2001 and 2006, the SNP differences ranged from four to 13.”

R1c2 Lines 294-295: With less than ≤18 SNPs, are they still termed “closely related strains”. In a �4 Mb genome sequence, what would be the NGS contribution to SNPs? Although this depends on the depth of sequencing, still there would be contribution from the sequencing itself.

Response: There isn’t a standard cutoff for SNP differences with respect to strain separation (Brown et al. 2019) (9). Parameters defined in the CFSAN pipeline for filtering the SNPs should minimize the contribution of NGS to SNPs. The CFSAN pipeline constructs a high-quality SNP matrix for closely related sequences (<100 SNP differences) with a higher recovery rate of SNPs for datasets with 100x coverage compared to 20x, as described by Davis et al., 2015 (12). Our genomes were sequenced with an average coverage of 50x.

We believe that modifications to this section (as described above) have addressed this question.

R1c3 Although AMR genes were identified from the WGS, these are not correlated with the antibiotic resistance phenotype of isolates.

Response: Throughout the section on AMR we specifically note the WGS refers to genotype and not phenotype. However, in the last paragraph of the “Antimicrobial resistance profile” section we discuss the information that is available on genotypic vs phenotypic expression of AMR genes in some of the isolates associated with the study (copied below). We believe this addresses the reviewer concerns and have not made changes to the manuscript.

“Antibiotic resistance profiles by the calibrated dichotomous sensitivity method were determined for Salmonella isolated from 2001 through 2005 but not for isolates from 2006, 2007, 2010, and 2013. Ten of the Salmonella survey isolates from 2001 to 2005 were resistant to three or more antibiotics [5]. Resistance genotype and phenotype correlated highly for five of these isolates: Salmonella Anatum (n = 3), Salmonella Istanbul (n = 1), and Salmonella Typhimurium var. Copenhagen (n = 1).”

R1c4 Only two pathogenicity islands (SPI-1 & SPI-2) were identified in WGS. What about the rest?

Response: SPI-1 and SPI-2 were present in all the isolates and we made note of this. Genes from other known pathogenicity islands were present but we did not assess the presence of complete islands in every isolate. The magnesium uptake genes (mgtB-mgtC) were present in all isolates (line 502) and are part of SPI-3. The hlyE hemolysin (lines 511) is part of SPI-18 and was identified in S. Horsham.

PLoS One. doi: 10.1371/journal.pone.0291109.r003

Decision Letter 1

Iddya Karunasagar

22 Aug 2023

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

PONE-D-23-14605R1

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PLoS One. doi: 10.1371/journal.pone.0291109.r004

Acceptance letter

Iddya Karunasagar

29 Aug 2023

PONE-D-23-14605R1

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

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

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(XLSX)

Click here for additional data file.^{(123.2KB, xlsx)}

Data Availability Statement

All the data are in the NCBI database with the following links: https://www.ncbi.nlm.nih.gov/bioproject/PRJNA941918 https://www.ncbi.nlm.nih.gov/bioproject/PRJNA951760

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PERMALINK

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

Anne-laure Moyne

Opeyemi U Lawal

Jeff Gauthier

Irena Kukavica-Ibrulj

Marianne Potvin

Lawrence Goodridge

Roger C Levesque

Linda J Harris

Roles

Abstract

Introduction

Materials and methods

Isolate selection

Table 1. Results of the WGS serotyping and number of plasmids for Salmonella survey isolates retrieved from raw almonds.

Whole genome sequencing

Quality control and genome assembly

Core genome SNP typing

Genetic distance

Virulence and antibiotic resistance (AMR) gene prediction

Plasmid detection and reconstruction

Results

Phylogenetic analysis of Salmonella isolates retrieved from raw almonds

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).

Genetic distance within each serovar

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

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

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

Plasmid prediction and characterization

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

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

Antimicrobial resistance profile

Analysis of virulence factors

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

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

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

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Iddya Karunasagar

Roles

Author response to Decision Letter 0

Decision Letter 1

Iddya Karunasagar

Roles

Acceptance letter

Iddya Karunasagar

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

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