Journal of Agriculture and Food Research 2 (2020) 100045

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Journal of Agriculture and Food Research

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Quantitative microbial risk assessment for Salmonella: Inclusion of whole

genome sequencing and genomic epidemiological studies, and advances in
the bioinformatics pipeline
Jinyao Chen a, c, Shraddha Karanth a, Abani K. Pradhan a, b, *
a
Department of Nutrition and Food Science, University of Maryland, College Park, MD, 20742, USA
b
Center for Food Safety and Security Systems, University of Maryland, College Park, MD, 20742, USA
c
West China School of Public Health, Sichuan University, Chengdu, Sichuan, China

A R T I C L E I N F O A B S T R A C T

Keywords: Despite the implementation of extensive measures to control food-borne illness caused by Salmonella since the
Salmonella beginning of the 20th century, it remains one of the most important and widespread food-borne pathogens.
Quantitative microbial risk assessment Quantitative microbial risk assessment (QMRA) has become essential to evaluating and mitigating the public
Whole genome sequencing
health risk of Salmonella. However, current QMRA models do not incorporate the minute changes in microbial
Food safety
Genomic epidemiology
behavior arising from differences in the microbial genome. Genomic analysis, particularly whole genome
sequencing (WGS), has undergone significant progress over the past two decades, facilitating rapid data accu-
mulation. This presents an unprecedented opportunity to incorporate genomic data into a QMRA for Salmonella.
This review aims to explore the applicability of WGS and other genomic technologies in Salmonella QMRA, and to
outline the added value and current limitations and promise of genomics, particularly bacterial WGS, in sup-
porting the integration of this data in a framework to assess the risk of Salmonella. Our review indicated that
although comparative genomic studies for Salmonella using WGS has seen a considerable amount of interest over
the past decade, the incorporation of genomic data into QMRA remains a difficult and arduous prospect. However,
with the progress in bioinformatics and genomic epidemiology, genomic data could certainly be utilized in QMRA
to improve and update risk assessment models and risk management strategies for Salmonella, as well as other
foodborne pathogens of interest.

1. Salmonella variance: Food safety implications
The implementation of several preventative and control measures
against Salmonella since the beginning of the 20th century has failed to
make a significant impact on its worldwide prevalence rates. Salmonella
is a major food-borne pathogen, with high morbidity and mortality rates
and demonstrated major economic loss worldwide [1–3]. According to
the World Health Organization (WHO), as of 2015, non-typhoidal Sal-
monella is considered to be 1 of the 4 key global causes of diarrheal
diseases, and affects an estimated 550 million people worldwide every
year, including 220 million children under the age of 5 years. This report
estimated the global impact of Salmonella to be approximately 31–211
million illnesses and 36,341–89,045 deaths, with a disease burden of
2.4–6.2 million disability-adjusted life years (DALYs) [4]. Globally, Sal-
monella prevalence and survival has been identified and reported in
several heterogeneous environments; consumable animal sources, spe-
cifically chicken and eggs, have been reported as major sources of human
Salmonella infection [5–7]. However, recent outbreak data have also
identified vegetables, nuts, and fruits, such as melons and papayas, as
possible novel routes for human infection [5,8,9].
The genus Salmonella is composed of two distinct species: S. bongori
and S. enterica, the latter of which is divided into six subspecies. These
subspecies have been classified into more than 50 serogroups and 2500
serovars based on the O (somatic) and H (flagellar) antigens [10].
Although different serotypes do not actually imply pathogenicity, a
limited number of serotypes have been associated with a majority of the
cases of human infections. Among these, Salmonella enterica serovar
Enteritidis (referred to hereafter as S. Enteritidis) is the most frequently
detected serovar, with incidence rates of 39.5% and 14.5% in the EU (in
2013) and the U.S. (in 2012), respectively, followed by Salmonella

* Corresponding author. Department of Nutrition and Food Science, University of Maryland, 0112 Skinner Building College Park, MD, 20742, USA.
E-mail address: akp@umd.edu (A.K. Pradhan).

https://doi.org/10.1016/j.jafr.2020.100045
Received 30 December 2019; Received in revised form 23 March 2020; Accepted 23 April 2020
2666-1543/© 2020 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
J. Chen et al.
enterica serovar Typhimurium (referred to as S. Typhimurium hereafter)
(including its monophasic variant; incidence rates of 28.8% and 11.6% in
the EU and U.S. in 2013 and 2012, respectively) [11,12]. S. Enteritidis is
commonly associated with poultry and its products, whereas S. Typhi-
murium has been isolated from a wider range of species, including pigs
and cattle, in addition to poultry [13]. However, recently, several Sal-
monella serovars other than the two considered to be major players in the
food safety domain (S. Typhimurium and Enteritidis) have been more
frequently implicated in food-borne outbreaks. For example, an unusual
serovar of Salmonella (ultimately recognized and named as S. Stanley)
was identified as the causative organism of a cross-border outbreak
(comprising 10 European Union countries) with over 700 non-travel
related human cases that occurred between 2011–2012 [13]. Similarly,
there has been an increase in the incidence of multi-state outbreaks
linked to S. enterica serovars Montevideo, Infantis, Javiana, Braenderup,
Adelaide, and Heidelberg, and other unusual serovars in the U.S [14–17].
2. Diversity in Salmonella virulence and pathogenicity profiles
All serovars belonging to Salmonella enterica share a common genome
structure, and the same core genome [10,18,19]. For instance, the
serovars Typhi and Typhimurium have 11% difference in their genome,
which are otherwise 99% similar in their sequences of housekeeping
genes [10,15]. However, there is enormous variation in the virulence,
host range, and epidemiology of different serotypes of Salmonella. For
example, only specific serotypes (S. Typhi, S. Paratyphi A and C, and S.
Sendai) cause enteric fever, while most serotypes cause only gastroen-
teritis. Similarly, a few other non-typhoidal serovars such as S. Choler-
aesuis and S. Dublin are more likely to cause bacteremia than diarrhea
[20,21]. Furthermore, some serovars are host adaptive while others are
more host-specific [22,23].
Salmonella pathogenicity islands (SPIs) are DNA segments encoding
virulence genes that are absent from the corresponding region of the
Escherichia coli K12 genome sequence. These gene-clusters are thought to
have been acquired by Salmonella from other species through horizontal
gene transfer [24]. The presence of virulence determinants such as type
III secretion systems (T3SS) allows Salmonella to be intracellular patho-
gens, distinguishing them from commensal E. coli [10,24]. Salmonella
undertakes several evolutionary measures, including gene acquisition
through horizontal transfer (associated with SPIs, transposable elements,
phages, and plasmids) and the loss of genes or a loss of their function,
which impacts host range. The latter might be the case with typhoidal
serovars: comparative molecular analyses have suggested that the ge-
nomes of the human-specific serovars Typhi and Paratyphi A show
considerable genome degradation and the accumulation of pseudogenes,
while being characterized by a unique complement of virulence de-
terminants, including the type IVb pilus operon [25,26].
So-called virulence factors determine the virulence of bacterial
strains. Actually, terms such as gene virulence and virulence factors are
not strictly defined [20,27,28]. In the case of Salmonella, the list of
candidate virulence factors increases with the ongoing increase in
knowledge on the molecular mechanisms underlying its pathogenicity.
Virulence factors for Salmonella now include virulence plasmids, which
are genes encoding toxins, fimbriae, flagella, antimicrobial resistance,
and other factors essential for pathogenicity [24,27,29]. So far, 21 SPIs
have been identified for Salmonella, and the continuous process of evo-
lution may add more to this list.
SPI-1~SPI-5 are classic pathogenicity islands that are commonly
shared by a majority of the serovars of Salmonella. SPI-1 contains a
number of functional genes, and is therefore responsible for the cyto-
toxicity of macrophages, invasion of epithelial cells, inflammation and
fluid secretion in the ileum, and cytokine secretion, thereby playing a
pivotal role in both gastroenteritis and systemic infection [30,31]. Genes
within the SPI-2 region are not essential for gastroenteritis but are
indispensable for systemic infection, as they support the intracellular
survival of bacteria within host cells [32]. Type-III secretion systems 1
2
Journal of Agriculture and Food Research 2 (2020) 100045
and 2 (T3SS-1 and T3SS-2) are encoded within SPI-1 and SPI-2, respec-
tively, and it has been proven that deletion of the SPI-1 or SPI-2 regions
can abrogate the virulence and ability of Salmonella to invade, colonize,
or replicate within the host cells [30,33]. On the other hand, SPI-3 en-
codes mgtC, which enables Salmonella survival under Mg2þ starvation
conditions and is therefore required for survival within macrophages, as
well as to induce systemic infection in mouse cells [24,30]. SPI-4 encodes
a type I secretion system (T1SS) and mediates adhesion, whereas SPI-5
includes SopB, which encodes the phosphatase portion of T3SS-1 [34].
While little information is available about the other SPI, studies have
shown that SPI-7 is exclusively present in the host-specific S. Typhi and
absent in S. Typhimurium [35], and SPI-8, -9, and -10 encode a pseudo
bacteriocin and degenerate integrase, a type I secretion system like SPI-4,
and a chaperone usher fimbrial operon, respectively [30,35,36].
Salmonella attachment to the target cell occurs over several steps
mediated by various adhesins encoded by fimbrial genes, such as type 1
fimbriae (fim), plasmid encoded fimbriae (pef), long polar fimbriae (lpf),
or thin aggregative fimbriae (Agf). Virulence plasmids, which contain the
7.8-kb Salmonella plasmid virulence (spv) locus, also play a significant
role in the virulence of Salmonella. However, virulence plasmids show a
remarkable diversity in the combination of virulence factors they encode,
which appears to support their host specificity and ability to cause
gastroenteritis or systemic disease [37,38].
It is common knowledge that effector proteins encoded by a complex
network of functional virulence genes allow Salmonella to survive under
various conditions effected by immune cells such as macrophages, neu-
trophils, and dendritic cells. Specific virulence determinants may be
clustered together on polymorphic pathogenicity islands or located on
transmissible genetic elements such as plasmids or phages that facilitate
the transmission of genes involved in pathogenesis, thereby increasing
the diversity in virulence profile among strains [30,39,40]. Based on
recent findings that multiple individual virulence genes have a variable
distribution in Salmonella, it would be safe to conclude that a complicated
combination of genes contribute to the overall virulence diversity, which
presents a big challenge for virulence profiling and, by extension, for risk
assessment.
3. Whole genome sequencing (WGS) in microbiology:
Advantages and limitations in food safety
Laboratory identification and differentiation of bacterial infectious
agents are critical for timely detection of foodborne outbreaks, identifi-
cation of the transmission vehicles, and retraction of contaminated food
from circulation [41]. Investigations into a foodborne outbreak relies on
the ability to subtype the etiological agent to differentiate it from cases
caused by non-related agents. Moreover, possible co-circulation of several
strains of foodborne pathogens among the population could confound
epidemiologic investigations [42]. In this regard, the emerging WGS-based
techniques, which provide complete data to enable the elucidation of
phylogenetic relationships and monitoring of disease-causing lineages
over time, offer a resolution and evolutionary context far exceeding that of
traditional phenotypic molecular subtyping methods [43].
WGS bypasses the need for a genetic map by using bacterial clones to
produce a large amount of redundant sequence read data across the
genome and utilizing computational technology to assemble the
sequence reads; subsequently, WGS has become the most popular and
advanced sequencing method in the last decade [44,45]. More recently,
high-throughput next-generation sequencing technology has paved the
way for cost-efficient and rapid WGS which can be used for real-time
detection [46]. Before the maturation of WGS, different DNA profiling
techniques such as multiple-locus variable number of tandem repeat
analysis (MLVA), pulsed-field gel electrophoresis (PFGE), and multi-locus
sequence typing (MLST) were found to have drawbacks such as diffi-
culties in standardization and comparatively lower resolution [47,48].
Moreover, WGS could greatly improve the speed, cost, analytical spec-
trum, and resolution of obtained data [49,50]. A study comparing the
J. Chen et al. Journal of Agriculture and Food Research 2 (2020) 100045

capability of various methods used to subtype S. Enteritidis revealed that
WGS resolved all outbreak clusters and provided robust inference results
with higher epidemiological correlation compared to PFGE and MLVA;
moreover, WGS was shown to cluster regularly interspaced short palin-
dromic repeats in combination with multi-virulence-locus sequence
typing (CRISPR-MVLST) [41], showing the high applicability of this
method in molecular subtyping of complex gene sequences.
With the realization of WGS in microbiology, the robust and higher
resolution genomic data provided has yielded important insights into the
transmission pathways of several significant pathogens including Sal-
monella. For example, WGS has been utilized in several recent epidemi-
ological investigations for microbial source attribution, in
microbiologically linking current outbreaks with historic cases of food-
borne illness, in differentiating outbreak cases with sporadic cases of
illness, and most recently, in geographic source attribution [42,51–59].
However, the challenge of converting primary sequence data into useful
public health action, in the form of microbial risk assessments and pre-
diction of antimicrobial resistance patterns, remains primarily unmet.
This could be due to the lack of a comprehensive bioinformatics pipeline
and information technology (IT) support to analyze the data and obtain
the specific information required for modeling and bacterial feature
prediction [60–62].
4. Databases and initiatives for Salmonella genomic analyses
Currently, the bottleneck in implementing WGS for public health
purposes has shifted to post-sequencing bioinformatics analysis of WGS
data. The failure to properly harmonize and interpret available WGS data
will lead to data fragmentation to the point that phenotypic traits such as
strain pathogenicity may be regarded as being different for different
strains due to the use of distinct approaches of sequence assembly and
annotation [12]. Moreover, the WGS revolution has resulted in the
identification of a large number of genes with unknown function to date;
therefore, gene annotation and functional evaluation should be con-
ducted concurrently with sequencing. Another factor to be considered in
the post-sequencing bioinformatics process is that WGS and mathemat-
ical modeling appear to show opposing tendencies, one towards greater
detail and better distinctions and the other towards simplicity and gen-
erality [63].
Several public repositories of sequencing data for Salmonella are
currently available, with published sequences being made available for
comparative genomic analysis. Table 1 lists several databases and ini-
tiatives for Salmonella genomic analyses, including the National Center
for Biotechnology Information's (NCBI) GenBank database, the U.S. FDA
GenomeTrakr network, the NCBI Pathogen Detection database, the
Collaborative Management Platform for Detection and Analyses of (Re)-
Emerging and Foodborne Outbreaks in Europe (COMPARE), and the
Global Microbial Identifier (GMI), among others.
Additionally, there are a number of online resources for species-
specific typing and subtyping, such as the Salmonella In-Silico Typing
Resource (SISTR; https://lfz.corefacility.ca/sistr-app/), the direct iden-
tification of specific pathogenic genes from assembled genomes, as the

Table 1
Several databases and initiatives for Salmonella whole genomic analyses.
Database Website Contributors/organizers/initiators Purpose

National Center for Biotechnology
Information's (NCBI) GenBank
database
GenomeTrakr
Collaborative Management Platform
for Detection and Analyses of (Re-)
Emerging and Foodborne Outbreaks
in Europe (COMPARE)
Global Microbial Identifier (GMI)
Pathosystems Resource Integration
Center (PATRIC)
Center for Genomic Epidemiology
https://www.ncbi.nlm.nih United States Food and Drug Administration's (U.S. Lists over 10,935 complete (and annotated) genome
.gov/genome/genomes/152 FDA) Center for Food Safety and Applied Nutrition assemblies of Salmonella uploaded by a worldwide
(CFSAN), the United States Department of network of labs, programs, and initiatives that are
Agriculture (USDA) Food Safety Inspection Service responsible for the generation of WGS data (and its
(FSIS), the U.S. Centers for Disease Control and related metadata).
Prevention (U.S. CDC), the CDC's National
Antimicrobial Resistance Monitoring System for
Enteric Bacteria (NARMS), the European Molecular
Biology Laboratory (EMBL), and the DNA Data
Bank of Japan (DDBJ), among others.
https://www.fda.gov/f U.S. FDA The first open-source network of its kind that
ood/whole-genome-seq utilizes WGS for pathogen identification. As of
uencing-wgs-program/g December 2019, GenomeTrakr participants include
enometrakr-network 15 federal laboratories, 25 state public health and
academic laboratories, 1 U.S. hospital lab, 2 other
labs located in the U.S., and 21 laboratories outside
the U.S., and several other laboratories, which are
authorized to collect WGS data and metadata of
foodborne pathogens, subsequently sharing them
via publicly accessible databases at the NCBI.
http://www.compare European Union's Horizon 2020 A large EU project comprising a multidisciplinary
-europe.eu/ research network of 29 European participants from
10 EU countries and Australia, with the aim of
speeding up the detection of, and response to,
disease outbreaks among humans and animals
worldwide through the use of new genome
technology.
http://www.globalmicr EU pilot program A network of approximately 160 representatives
obialidentifier.org/About from 32 countries working to develop a global
-GMI system to aggregate, share, mine, and use
microbiological genomic data to address global
public health and clinical challenges.
https://www.patricbrc. Wholly or in part funded by the U.S. National A genomics-centric relational database and source
org/portal/portal/pat Institute of Allergy and Infectious Diseases, U.S. for the analysis of bacterial gene expression data.
ric/Home National Institutes of Health, and the Department
of Health and Human Service
http://www.genom National Food Institute, Denmark Aims to provide the scientific foundation for a
icepidemiology.org/ future central database of genome data, a platform
of spatio-temporal tools for the analyses of such data
along with epidemiological information, as well as a
web-based interface capable of facilitating the
exchange of required microbial data.

3
J. Chen et al.
Antimicrobial Resistance Identification by Assembly (ARIBA; https://g
ithub.com/sanger-pathogens/ariba), or the identification of gene fam-
ilies that correlate with pathogenic phenotypes to distinguish them from
relatively harmless microorganisms, such as the PathogenFinder (htt
ps://cge.cbs.dtu.dk/services/PathogenFinder/) [64]. However, many of
these databases and tools do not contain sufficient metadata regarding
the date, time, or conditions under which the isolates were obtained to
make useful conclusions about the behavior of pathogens in food or in
their human hosts.
There are, however, publicly available databases to look into the
virulence genes of Salmonella, and their functionality. The virulence
factor database (VFDB) (http://www.mgc.ac.cn/VFs/) is a constantly
updated database of various virulence factors (and their corresponding
genetic sequences) present in pathogenic bacteria, including E. coli,
Salmonella, and Vibrio [65]. Pathogenicity Island Databases (PAIDB) is a
database dedicated to providing comprehensive information on all an-
notated and predicted pathogenicity islands (PAIs) in prokaryotic ge-
nomes, containing 223 types of PAIs with 1331 accessions [66].
Alternatively, the “Virulence Search” database (http://www.hp
a-bioinfotools.org.uk/pise/virfactfind_small.html), developed by Public
Health England, is an engine that was designed to predict the potential
virulence factors in unannotated genomes [67]. Most of the above
pathogen and pathogenicity prediction tools rely heavily on a structured
database of pathogens to inform their pathogenicity inference. This
severely hinders their performance on unknown, unrecognized, and un-
mapped sequences [68]. Recent approaches to analyzing and interpreting
WGS data has shifted towards the inference of pathogenic phenotypes
using a number of machine learning- and deep-learning-based tools.
Examples include the Pathogenicity Prediction for Bacterial Genomes
(PaPrBaG; https://github.com/crarlus/paprbag) [69], which trains on a
wide range of species with known pathogenicity phenotype to overcome
genetic divergence, and the Deep Learning Approach to Pathogenicity
Classification (DeePAC; https://gitlab.com/rki_bioinformatics/DeePaC)
[68], which infers pathogenic phenotypes without the aid of databases of
known pathogens.
The status of the above databases and programs suggests that despite
the rapid development of WGS and basic comparative genome analyses,
there is a significant lag in the interpretation of raw data in a phenotypic
context. Moreover, there are no standard procedures or experimental
protocols for the exploration of phenotypic characteristics. While some
components necessary for phenotypic prediction and comparison based
on WGS data have been developed, there currently exists no single in-
tegrated platform to provide this kind of analysis. Moreover, there is no
specific repository of widely applicable genomic and phenotypic analyses
for Salmonella, although a predictive genomic platform for E. coli
(https://lfz.corefacility.ca/superphy/) is under development [70].
Comparative genomic analyses of Salmonella would be a crucial first step
in the evolution of these platforms; this could help in establishing the
framework to pool genotypic and phenotypic information obtained by
Salmonella researchers worldwide.
5. Comparative genomic analysis of Salmonella
At this point, filtering genome sequences to locate genes relevant to
its virulence is an essential step towards integrating molecular data into
risk assessment. However, the physiological impact of the occurrence,
expression, and interaction of such genes poses a major challenge.
Despite the availability of several tools (both old-school and emerging)
for genome sequence analyses, data mining for virulence genes and
predicting their observable phenotypes is still in its infancy. Although
genetic analyses and comparisons are required, determining the pheno-
typic characteristics (such as adhesion, invasiveness of intestinal
epithelial cells, antimicrobial resistance, and animal virulence) encoded
in these genetic sequences by performing functional (proteomic) or
phenotypic assays in combination with gene expression analyses would
be more preferable. In the face of a lack of experimental phenotypic
4
Journal of Agriculture and Food Research 2 (2020) 100045
information, comparative genomic research could also support in-
vestigations into the molecular differences between isolates from
different hosts, types of food, Salmonella serovars, and geographical and
spatial locations, as well as the differences between serovars that could
lead to the manifestation of different types of disease. This could also aid
in the understanding of virulence genes and their combinations. To date,
comparative genomic analyses have been performed on various groups of
isolates of Salmonella, with the corresponding phenotypic traits being
assessed by comparative phenotypic or proteomic studies [15,21,71–73].
Therefore, gaining a thorough understanding of virulence genes and gene
profiles has been employed as the first step towards integrating Salmo-
nella WGS data with food safety risk assessment. With this in mind,
WGS-assisted comparative genomic studies published alongside (or not)
phenotypic analyses or proteomic studies of Salmonella were collected
and listed in Table 2; the studies were retrieved using various combi-
nations of the keywords “whole genome sequence”, “comparative
genomic study”, “virulence phenotype”, “proteomic analysis”, and “Sal-
monella.” Antimicrobial resistance genes were not considered at this
point.
Current efforts in the area of Salmonella genomics involve the use of
WGS, followed by comparative genome analysis, in elucidating the
evolutionary relationships between bacterial species. However, as the
evolution and phylogeny of the various Salmonella serovars was not the
primary focus of this literature review, only studies that investigated the
genetic variations in virulence determinants were included. Among the
studies included in Table 2, nine focused on genomic variety and
phenotypic diversity, six of which reported on a phenotypic analysis
coupled with WGS. All nine studies indicated the presence of consider-
able genomic variation within the Salmonella genus; in fact, some of these
studies confirmed that Salmonella Typhi and Paratyphi A, serovars
restricted to human hosts, characteristically displayed genome degra-
dation and pseudogene accumulation, as well as acquisition of horizon-
tally transferred gene elements [10,15]. Most studies concluded that
horizontal gene transfer occurs frequently among all Salmonella strains,
and that the composition of mobile genetic elements with virulent plas-
mids and phages varies within and across serovars (inter- or
intra-serovar) [10,14,15,20,21,71,72]. Two of the six studies that con-
ducted phenotypic analyses conducted mutant or knock-out tests for the
genes of interest. While one established the hmpA and katE genes as being
responsible for the loss of fitness under nitrosative and oxidative stress in
S. Typhimurium, which might be associated with host adaptation [15],
the other determined that the gene st313-td contributed to the intracel-
lular survival of S. Typhimurium in murine macrophages, and that its
deletion would attenuate the virulence of the isolates [73]. Among
studies that focused on the phenotypic and genotypic variations among
various Salmonella strains, three focused on metabolic traits such as the
utilization of different carbon sources [72], β-glucuronidase activity
[71], catalase activity, and growth patterns in the presence of tetrathi-
onate and under nitrosative stress [15]. Alternately, three other studies
looked directly into the virulent characteristics of various Salmonella
serovars [19,21,73] by analyzing their ability to infect mice, via in vitro
tests [21,73], and using a test to determine virulence against Caeno-
rhabditis elegans [19]. Noteworthy studies include those conducted by
Fricke et al. (2011) and den Bakker et al. (2011), wherein the phenotypic
variations in utilization of carbon substrates between genetically similar
Salmonella isolates and the effect of genes related to β-glucuronidase
activity on host adaption, respectively, were observed [71,72]. These
studies implied that phenotype-based risk assessment research should
focus on the metabolic aspect of various Salmonella serovars in addition
to their virulence characteristic. However, the above studies failed to
directly link the genotype and/or phenotype geographically and spatially
and to outbreak and sporadic cases. This is despite specific genes being
linked to increased virulence; examples of such genes include st313-td,
which is correlated with increased invasiveness of S. Typhimurium [73],
and SPI-2, whose defection was linked to the incompetence of S. Ken-
tucky [14].
J. Chen et al.
Table 2
Comparative genomic studies of Salmonella by WGS.
Authors, year, Description of the isolates
country
1 Jacobsen et al. 45 sequenced Salmonella genomes
(2011) [10], that were publicly available at the
Denmark time of the study.
2 Fricke et al. 17 non-typhoidal Salmonella strains
(2011) [72], (classified into 12 serovars) isolated
U.S. from human, animal, and food
sources.
3 den Bakker MLSA and SNP analysis of 16 newly
et al. (2011) sequenced and 30 publicly available
[71], U.S. genomes resulted in the division of
Salmonella into two clades, A and B.
An additional set of 123 isolates was
assigned to clades A and B by
targeting subpopulation-specific
genes and SNPs with qPCR.
4 Hayden et al. 114 S. Typhimurium strains
(2016) [15], collected between 1946 and 2012
U.S. from humans and food animals in
the U.S. and abroad.
5 Herrero- 295 S. Typhimurium and 50 S.
Fresno et al. Dublin isolates from 10 countries
(2014) [73], were analyzed for the presence of
Denmark st313-td. Six S. Typhimurium strains
from around the world were
sequenced.
6 Dhanani et al. 25 Salmonella isolates classified into
(2015) [14], 5 serovars from broiler chicken
Canada commercial farms in Canada; some
of the isolates included in “Diarra
et al. (2014) [85]”.
7 Blondel et al. 44 completed or in-progress
(2009) [111], Salmonella genomes from a public
Chile database.
Phenotypic analysis
N/A
The Biolog phenotype microarray
system, which allows the testing of
nearly 1,200 metabolic and chemical
sensitivity phenotypes, was used to
assay the utilization of different
carbon sources.
β-glucuronidase activity was
determined by growing the isolates
in EC MUG broth.
(1) Bacterial growth in the presence
of tetrathionate and nitrosative
stress.
(2) Mutation of hmpA and katE was
tested in strains that showed growth
under nitrosative and catalase
activity, respectively.
(1) Mouse infection; (2) epithelial
cell infection; (3) macrophage
infection; (4) knock-out and
complementation study: the gene
st313-td was cloned into wild type S.
Typhimurium as well as knocked out
in S. Typhimurium ST313 followed
by complementation.
N/A
N/A
5
Journal of Agriculture and Food Research 2 (2020) 100045
Main findings of the study Virulence genes specifically focused
on
(1) The core and pan-genome of SPI-6 and SPI-7 found only in serovar
Salmonella were estimated to S. Typhi; SPI-3 exists in several similar
contain around 2,800 and 10,000 but distinct versions.
gene families, respectively.
(2) Gene content is often, but not
uniformly, correlated to serotype.
(3) The SPIs were among genes that
showed variations; variations were
also observed in phage insertion
sites and transposable elements.
(1) The evolution of known
Salmonella sublineages is mediated
mostly by the loss of coding
sequences with known metabolic
functions and the acquisition of
horizontally transferred phage and
plasmid DNA.
(2) Phenotypic variations between
closely related Salmonella isolates
were observed for utilization of
carbon substrates.
(3) Phenotypes are frequently lost
and/or acquired in different
Salmonella sublineages and thus
correlate poorly with the
evolutionary relationships within
this species.
(1) Salmonella consists of at least All clade B isolates contained SPI-18
two subpopulations (clade A and and a cytolethal distending toxin islet.
clade B) that differ specifically in The combination of these two islands
genes involved in host and tissue was previously thought to be
tropism, and utilization of host- exclusive to typhoid-associated
specific carbon and nitrogen serovars.
sources, suggesting differences in
adhesion, colonization properties,
and metabolic capabilities.
(2) Presence of β-glucuronidase in
clade B isolates suggests an
adaptation of this clade to the
vertebrate gastrointestinal
environment.
(1) A phylogenetic tree of S. Strains closely related to strains
Typhymurium was constructed. associated with human
(2) In the U.S., salmonellosis is immunodeficiency virus (HIV)-
caused by diverse S. Typhimurium infected individuals in sub-Saharan
strains circulating worldwide. Africa were found. Phenotyping
(3) No specific link was found studies linked hmpA and katE variants
between strains or lineages and in subclade 1a specific to these strains
specific outbreaks or sporadic to loss of fitness under nitrosative and
events. oxidative stress, respectively.
The gene st313-td is mainly The apparently restricted presence of
associated with invasive strains, st313-td in human isolates suggests a
st313-td contributes to S. potential host specificity for this gene
Typhimurium intracellular survival as suggested for its carrier, S.
within murine macrophages, and Typhimurium ST313.
not needed for invasion of the
epithelial cells, and st313-td knock-
out was attenuated in virulence in a
systemic murine infection.
(1) The study isolates showed Conserved T3SS and T4SS could be
variations in genes coding for candidates for pathogenicity
adhesins, flagella, iron acquisition, prediction.
T3SS, T4SS, and drug resistance, The lack of SPI-2-associated genes
suggesting the diversity of SopD2, PipB2, SspH2, and SrfH in
Salmonella serovars found in broiler isolates of S. Kentucky might explain
chicken. in part their inability to induce
(2) Existence of mobile genetic diseases in humans.
elements indicates that isolates
from a given serovar could acquire
and transfer genetic elements.
(1) The Salmonella T6SS loci were Data indicate that T6SS is widely
acquired by independent lateral distributed in some serovars (such as
transfer events and evolved to S. Dublin) and absent in serovars
(continued on next page)
J. Chen et al. Journal of Agriculture and Food Research 2 (2020) 100045

Table 2 (continued )
Authors, year, Description of the isolates Phenotypic analysis Main findings of the study Virulence genes specifically focused
country on

Three representatives of each branch
in previously described T6SS
phylogenetic trees were used as
baits.
8 Marzel et al. A case-control study of 48345
(2016) [21], culture-confirmed non-typhoidal
Israel Salmonella infections that occurred
in Israel between 1995 and 2012.
WGS data was used to analyze 22 S.
Typhimurium isolates from 11
patients with persistent, recurring
cases of salmonellosis.
9 Hoffmann 44 S. Heidelberg isolates including
et al. (2014) 30 with nearly indistinguishable
[19], U.S. PFGE patterns from various sources
between 1982 and 2011. This study
included nine isolates associated
with the 2011 S. Heidelberg
outbreak in the U.S.
The 22 S. Typhimurium strains were
tested for (1) growth in minimal
media; (2) biofilm formation; (3)
motility; (4) and replication within
macrophages.
Four pairs of isolates that showed
genetic and phenotypic differences in
vitro were chosen to perform a
streptomycin pre-treated mouse
colitis model test.
Caenorhabditis elegans virulence
assay.
contribute to the adaptation of Enteritidis, Gallinarum, Agona,
various serotypes to different Javiana, and Paratyphi B, among
lifestyles and environments, others.
including animal hosts. However,
these were not essential for
virulence.
(2) The authors identified 3 novel
T6SS loci, designated as SPI-19,
SPI-20, and SPI-21.
(1) A different composition of
mobile genetic elements (plasmids
and phages) or SNPs in global
regulators was identified in
multiple cases.
(2) Four NTS serovars (Mbandaka,
Bredeney, Infantis and Virchow)
were significantly more frequently
associated with persistence than
others in this study.
(3) Though clear directionality of
neither genetic nor phenotypic
changes between early and later
same-patient isolates were
identified, differences in phenotype
and virulence between early- and
late-stage same-patient isolates
were found, specifically in-host
microevolution and its potential to
shape Salmonella pathogenicity.
(1) Substantial loss and gain of 107 previously characterized
plasmids and phages between these virulence genes (84 pathogenicity and
S. Heidelberg isolates that were 23 fimbrial markers) and plasmids:
consistent with previously SPI-1~5, the lambda-like prophage
generated tree topologies, Gifsy-2, and/or islets, T3SS, sopE, and
suggesting rapid exchange of these adhesins were present in all S.
plasmids. Heidelberg isolates. On the other
(2) S. Heidelberg isolates carrying hand, SPI-7, prophage Gifsy-1, Gifsy-
components of the T4SS were more 3, Fels-1, and spv were not identified
pathogenic in C. elegans model than in any of the 44 isolates.
those not carrying the system,
indicating T4SS plays a role in the
pathogenicity of S. Heidelberg.

SPI – Salmonella pathogenicity islands.

NTS – non-typhoidal Salmonella.
PFGE – pulsed-field gel electrophoresis.
TxSS – Type-x secretion system (where x denotes the number; for example, T4SS).

Undoubtedly, the high discriminatory capacity of WGS has cemented
its position as the new gold-standard method for comparative genomic
studies of isolates. However, since a search of literature pertaining to
comparative genomic studies using WGS provided us with a limited
number of studies, we expanded our search to other comparative
genomic studies carried out by comparative genomic hybridization
(CGH; Table 3), as well as to studies that investigated multiple virulence
factors and phenotypic traits using polymerase chain reaction (PCR;
Table 4). Microarrays comprising multiple genes from the genome of a
typical Salmonella serovar such as S. Typhimurium LT2 or S. Typhi CT18,
those combined with specific genes from other serovars, as well as
microarrays comprising genes from the Salmonella pan-genome are
available commercially [25,40,74]. However, the results of CGH and PCR
using these microarrays should be interpreted with caution as
microarray-based analyses cannot detect mutated genes, or those that
have not yet been identified by sequencing [75].
Fourteen CGH studies focusing on virulence gene expression and
phenotype comparison and eleven studies analyzing multiple virulence
factors using PCR (since 2000) were included in this review. The results
of the CGH studies corroborated those of the WGS studies with respect to
the variability in the virulence, potency, and survival characteristics of
Salmonella. Pan et al. (2009) found differences in the genes, and the
corresponding phenotype, coding for the plasmid-borne pef operon and
amino acid transportation genes, which are responsible for nitrogen
utilization, epithelial cell invasion, and biofilm formation, between the
prevalent PTs 4, 6, 8, and 13a and non-prevalent PT11 strains [74].
Therefore, these genes could be of considerable interest as targets in
future studies. On the other hand, Betancor et al. (2009) [39] and Yim
et al. (2010) [76] reported that phage SE20 and phage-associated genes
might contribute to the epidemiological nature of S. Enteriditis. In fact,
Betancor's study detected major differences in the in vitro infection ability
with strains with indistinguishable genetic patterns being based on the
Salmonella pan-genome [39], which reflects the considerable variability
in the Salmonella genotype and phenotype, and highlights the need for
high-resolution WGS as comparative tool for Salmonella identification
and characterization.
Studies conducted after 2000 using traditional PCR primarily focused
on specific virulence genes in Salmonella. However, as the analyzed iso-
lates were obtained from different sources present in the same
geographic area, a majority of these studies could not identify specific
source-related or prevalence-related virulence profiles. Among these
studies, phenotypic analyses were performed in only two studies: one
study conducted using a mouse lethality assay [77] reported a conflicting
finding that outbreak strains may be avirulent, while the other, which

6
J. Chen et al.
Table 3
Comparative genomic studies of Salmonella by Comparative genomic hybridization (CGH) with or without other methods.
Authors, Description of the isolates
year, country
1 Betancor 266 S. Enteritidis isolates
et al. (2009) from different sources.
[39],
Uruguay
2 Yim et al. The 29 isolates studied in
(2010) [76], “Betancor et al. (2009)
Uruguay [39]” 15 isolates from
human gastroenteritis, 5
from invasive disease, and
9 from nonhuman sources
in Uruguay.
3 Pan et al. 37 S. Enteritidis strains
(2009) [74], from five phage types
UK (prevalent and non-
prevalent) from different
sources in UK.
4 Morales et al. A field isolate of S.
(2005) Enteritidis PT4, a wild-type
[112], U.S. S. Enteritidis PT13A strain,
and a biofilm-forming S.
Enteritidis PT13A.
5 Anjum et al. 40 clinical and field
(2005) [18], Salmonella isolates from 12
UK common serovars.
6 Porwollik 22 S. enterica and 1
et al. (2002) S. bongori.
[79], U.S.
7 Porwollik 79 isolates from common
et al. (2004) prevalent serovars
[80], U.S.
8 Figueiredo 106 Salmonella strains from
et al. (2015) poultry (n ¼ 39), swine
[25], UK (n ¼ 14), cattle (n ¼ 1), and
processed food (meat cuts,
CGH used, and the
microarray description
The 266 S. Enteritidis
isolates were genotyped
using RAPD-PCR.
29 epidemic-spanning S.
Enteritidis field isolates
were subjected to CGH
using a Salmonella pan-array
based on the S. Typhi CT18
genome, complemented
with strain-specific genes
from other strains.
The CGH design and results
were the same as in
“Betancor et al. (2009)
[39]”.
CGH with a Salmonella pan-
genome microarray
covering seven Salmonella
genomes accompanied with
Pulsed field gel
electrophoresis (PFGE) and
multi-locus sequence typing
(MLST).
CGH with a DNA microarray
developed from sequence of
S. Enteritidis PT4
accompanied with PCR.
CGH with a DNA microarray
developed based on S. Typhi
CT18 chromosome.
CGH with a DNA microarray
developed based on S.
Typhimurium LT2 genome.
A microarray based on S.
Typhimurium LT2
supplemented with
annotated chromosomal
ORFs from the serovar
Typhi CT18, also containing
LT2 virulence plasmid pSLT
and R46
A customized Salmonella
virulence gene microarray
targeting 114 Salmonella
virulence determinants
present on the chromosome
Phenotypic analysis
Human epithelial cell
invasion analysis was
performed for the 29
isolates, spanning different
periods, genetic profiles,
and sources of isolation.
(1) Motility tests; (2) Caco-
2 adhesion and invasion
assays; (3) Bacterial
survival in egg albumen;
(4) Chicken virulence
assays.
(1) Epithelial cell adhesion
and invasion assays.
(2) Bio-forming assay and
colony morphology.
(3) Phenotype microarray
for nitrogen utilization and
osmotic stress.
(4) Inactivation of amino
acid transporter genes and
complementation.
Phenotype microarrays
assay the growth of
bacteria by measurement
of respiratory activity
under variable culture
conditions.
(1) Cells distention/cells
apoptosis analysis
(2) epithelial cells
adhesion/invasion assays.
(3) Galleria mellonella
7
Journal of Agriculture and Food Research 2 (2020) 100045
Main findings of the study
(1) 233 chromosomal genes
coding for metabolism and
bacterial fitness, and the
virulence plasmid analyzed
were found as variable among
S. Enteritidis isolates.
(2) Strains with
indistinguishable genetic
patterns still showed major
differences in their infection
ability; the approach used
was insufficient to detect the
genetic basis of differential
behavior.
(1) A great heterogeneity of
phenotypes was found, with
isolates belonging to identical
genetic profiles showing very
dissimilar behaviors.
(2) The pre-epidemic isolates
showed low levels of fitness in
most of the assays evaluated.
(3) The subset of isolates from
human gastroenteritis
showed higher homogeneity
of phenotypes and higher
levels of fitness than isolates
from any other sources.
Genetic and corresponding
phenotypic differences exist
between strains of the
prevalent PTs 4, 6, 8 and 13a
and non-prevalent PT11
strains: (1) strains from
prevalent PTs were less
susceptible to urea stress and
utilized amino acids more
efficiently than PT11 strains.
(2) in epithelial cell
association assays, PT11
strains were less invasive than
other prevalent PTs.
(3) Most strains from
prevalent PTs were better
biofilm formers than PT11.
It is not known how the
results of phenotype
microarray analysis correlate
with virulence genes
expression.
The scale of gene flux among
and within the various strains
is significant.
Gene contents sometimes
differed more within a
serovar than between
serovars.
Isolates of the same serotype
usually harbor the same
virulence profile.
Virulence genes specifically
focused on
Phages play a crucial role in
the generation of genetic
diversity in S. Enteritidis.
Phage SE20 may be a key
marker for the emergence of
particular isolates capable of
causing epidemics.
Loss of several phage-
associated genes improved
the virulence of S. Enteritidis,
rendering strains more prone
to cause an epidemic.
400 genes that differentiated
prevalent (4, 6, 8 and 13a)
and sporadic (11) PTs, of
which 35 were unique to
prevalent PTs, including six
plasmid-borne genes and four
chromosomal genes encoding
putative amino acid
transporters.
A single band shift within a
phage type may indicate an
evolutionary event in this
pathogen that has potential
for increasing human disease.
Most genes within SPI-1 to
SPI-5 and SPI-6, SPI-9, and
three fimbrial operons (fim,
bcf, and stb) were conserved
within all Salmonella strains
included in this study.
Outer membrane proteins
(Sops) is variable, and SPI-
1~5 is quite homologues.
The entire plasmid of pSLT
including spv is present in all
S. Typhimurium strains
included in this study.
(1) Virulence determinants
located in SPIs 1–6 and
adhesion were highly
conserved; prophage genes
shows great variability; genes
(continued on next page)
J. Chen et al.
Table 3 (continued )
Authors, Description of the isolates
year, country
hamburgers or sausages)
(n ¼ 52).
9 Zou et al. 24 Salmonella isolates
(2011) [81], including Roma tomato
U.S. outbreak strains, and
isolates from turkey farms
and chicken eggs.
10 Huehn et al. 523 Salmonella strains (S.
(2010) [84], Enteritidis, Typhimurium,
Europe Infantis, Virchow, and
Hadar) collected from
various host sources and
public health and
veterinary institutes across
nine European countries.
11 Suez et al. Low-passage clinical strains
(2013) [83], isolated from blood
Israel (n ¼ 66), stool (n ¼ 68) or
unknown source (N ¼ 1)
from 12 non-typhoidal
Salmonella (NTS) serovars
obtained from the Israeli
National Salmonella
reference Center.
12 Litrup et al. 58 Salmonella isolates from
(2010) [26], human faces and 10 from
Denmark human blood, and pigs
(n ¼ 3), cattle (n ¼ 1) and
hedgehog (n ¼ 1).
13 Reen et al. 12 S. Dublin, Agona, and
(2005) [86], Typhimurium isolates
Ireland recovered from bovine and
milk filter samples in
southern Ireland, and 2
clinical isolates from
CGH used, and the
microarray description
(n ¼ 90; SPIs, n ¼ 52, and
other chromosomal genes,
n ¼ 38), phage related
(n ¼ 12), and plasmid
related (n ¼ 12).
WGS for S. Infantis Sal147
(absence of SPI-1 genes),
and S. Typhimurium Sal199
(presence of cdtB of toxin);
PCR of SPI-11 genes and
typhoid-associated
virulence genes.
CGH composing of 69 gene-
specific oligonucleotide
probes including (SPI-1 to
SPI-5) virulence-associated
genes using S. Typhimurium
LT2 genome NC_003197 as
the reference.
PCR for 10 potential
virulence factors (avrA,
ssaQ, mgtC, siiD, sopB, gipA,
sodC1, sopE1, spvC, and
bcfC) for 523 isolates.
A representative subset of
77 strains was investigated
using a DNA microarray
designed to detect 102
virulence and 49 resistance
determinants.
CGH using a Salmonella
microarray (the STv7E
Salmonella microarray).
A DNA micro-array
containing 280 probes
covering genes related to
pathogenicity, phages,
antimicrobial resistance,
fimbria, mobility, serovars,
and metabolism with MLST.
The S. Typhimurium LT2
microarray, the same with
“Porwollik et al., 2004”,
including SPI1~SPI5 and
pSLT.
Phenotypic analysis
infection model, was
detected for S. Infantis
Sal147, Sal199, Sal280,
and S. Typhimurium LT2.
(1) The pathogenicity of
invasive isolates
representing the 12
serovars was investigated
in the mouse model.
(2) Virulence-associated
phenotypes were
compared between
invasive and
gastroenteritis isolates in
epithelial cells lines and
macrophages.
8
Journal of Agriculture and Food Research 2 (2020) 100045
Main findings of the study
58% of the virulence-
associated genes (40/69)
were present in all 24
Salmonella isolates tested
while the remaining genes
were variable in their
distribution.
(1) The virulotype did not
vary significantly with host
source or geographical
location.
(2) The highest variation was
observed in genes located on
prophages. Each serovar
demonstrated its own specific
fimbrial profile.
(1) A substantial degree of
intra-serovar variation in
virulence-associated
phenotypes was identified.
(2) Not all previous identified
virulence factors are
necessary for invasiveness.
(3) Intrigue results were
found that blood isolates were
not generally more invasive
than stool isolates of the same
serovar, and superior
intraepithelial cell replication
of gastroenteritis isolates
compared to invasive isolates
was observed in some
serovars.
(1) Differences in content of
pathogenicity related genes
between serovars with
various host preferences and
pathogenicity towards
humans were not observed.
(2) Except for 4 outbreak
strains of S. 4,5,12:b: the
characterization of strains
based on pathogenicity
related genes fits well with
the serovar assignment.
Only limited intraserovar
diversity was found among
the nine S. Dublin, three S.
Agona, and six S.
Typhimurium isolates.
Virulence genes specifically
focused on
in virulence plasmid are
present in all S. Enteritidis
and part of S. Typhimurium.
(2) S. Infantis Sal147, was
unable to invade HT-29 cells,
unlike Sal280, virulence was
decreased corresponding to
the absence of SPI-1 genes.
(3) The increased virulence
and cytotoxic effect by in vitro
cells apoptosis analysis and in
vivo mortality of G. mellonella
by S. Typhimurium Sal199
with respect to S.
Typhimurium LT2 might be
associated with expression of
the CdtB toxin genes.
Regardless of the serotype,
Salmonella isolates exhibited
most variability among the
iacP, avrA, sopE, sirA and invH
genes belonging to the SPI-1
class.
(1) Virulence determinants
located in the SPIs 1–5 were
highly conserved among the
five serovars except for SPI-3,
virulence genes in phage and
plasmid were variable.
(2) Some genes for
pathogenicity, e.g. spv, were
only found in S. Enteritidis,
Typhimurium.
(1) SPI 1–5, 9, 13, 14; five
fimbrial operons (bcf, csg, stb,
sth, sti); three colonization
factors (misL, bapA, sinH);
and the invasion gene, pagN
were conserved in all isolates.
(2) Except that tcfA that was
found to be more frequently
associated with invasive
isolates of S. Schwarzengrund
than with gastroenteritis
isolates, the study did not
identify a clear source-related
distribution of virulence
genes.
(3) Various NTS virulence
factors and six typhoid-
associated virulence genes
displaying a wider
distribution among NTS than
was previously known.
Strains from the supposedly
less virulent serovar S. Derby
lacked a combination of
virulence genes (truncated
SPI-3, irsA, msgA, and some
prophage genes).
SPI1~SPI5 were present in
all isolates. Plasmid pSLT was
absent from all S. Agona
isolates, and only
homologues of the spv genes
were present in eight of the
nine S. Dublin strains, and
(continued on next page)
J. Chen et al.
Table 3 (continued )
Authors, Description of the isolates CGH used, and the
year, country microarray description
Canada and 4 archival
isolates.
14 Beutlich 38 Salmonella strains PCR (10 virulence genes:
et al. (2011) isolated between 2002 and avrA, bcfC, gipA, mgtC, ssaQ,
[113], 2006 in eight European sopB, sopC1, sopE1, spvC, and
Europe countries from humans, spi4_D).
animals, and food. 9 representative Salmonella
isolates were analyzed by
DNA microarray (101 genes
including SPIs, prophages,
plasmids, islets, and fimbrial
clusters).
PFGE – Pulsed field gel electrophoresis.
MLST – Multi-locus sequence typing.
RAPD-PCR – Random amplification of polymorphic DNA polymerase chain reaction.
NTS – Non-typhoidal Salmonella.
SPI – Salmonella pathogenicity islands.
analyzed the production of aerobactin and colicin in various Salmonella
serovars [78], reported a difference in the production of the siderophore
and bacteriocin between the Typhimurim and Kentucky serovars.
Several CGH studies focused on the construction of a Salmonella pan-
genome, with the intention of locating the core and variable genes in the
genome, which are not very well defined [18,79,80]. For example,
although a majority of studies suggested that genes in the SPI-1~SPI-6
region were conserved, Zou et al. (2011) reported significant variability
among genes belonging to the SPI-1 class irrespective of the serotype in
twenty-four Salmonella isolates [81]. This defect in the genes belonging
to the SPI-1 class was also reported by Figueiredo et al. (2015) and
Majtan et al. (2007) [25,82] Additionally, Figueiredo et al. (2015) re-
ported that S. Infantis Sal147 displayed decreased invasiveness in the
absence of the SPI-1 region [25]. Studies conducted with Salmonella
serovars obtained from different geographical sources such as the U.S.
[71], UK [25], and Israel [83] suggested that the presence of
typhoid-associated virulence genes was not exclusive to typhoid-related
serovars; in fact, these studies concluded that these genes displayed a
wider distribution among non-typhoidal Salmonella strains than was
previously known, which brought to the fore questions regarding the
pathogenic mechanism of Salmonella as well as the predictability of
previous in vitro and even in vivo invasion assays. Correspondingly,
clinical trials conducted by Suez et al. (2013) obtained interesting results
about the phenotypic/epidemiological traits, such as the lower level of
invasiveness of the same serovar in blood isolates compared to stool
isolates and the greater fitness of gastroenteritis isolates compared to
invasive isolates grouped into some serovars [83].
The results of studies conducted by Zou et al. (2011) [81] using 24
Salmonella strains from the U.S., Litrup et al. (2010) [26] with 73 isolates
from Denmark, and Huehn et al. (2010) [84] using a larger sample of 523
Salmonella strains obtained from across Europe indicated that strains
belonging to the same serovar could be grouped together via virulence
profiling. This indicated that the broader virulence-associated gene
complemented the serovar classification. However, as these studies used
microarrays that were only focused on known virulence factors, the
significance of the former statement and the extent of correlation be-
tween the two should be further investigated using WGS.
Mobile genetic elements such as plasmid genes have generally shown
greater variability in distribution among strains compared to phage genes.
Moreover, the results of several studies have shown that genes occurring in
the plasmid (such as spv) are more frequently observed in the common
Salmonella serovars such as Enteritidis and Typhimurium [80,84–86]. This
conforms to the established idea that virulence plasmids are usually
Journal of Agriculture and Food Research 2 (2020) 100045
Phenotypic analysis Main findings of the study Virulence genes specifically
focused on
present in the six S.
Typhimurium isolates.
(1) Only minor serovar-
specific differences among
virulence patterns were
detected.
(2) Some virulence patterns
were serovar specific, e.g., all
S. Newport and most S.
Typhimurium strains shared a
common virulotype.
(3) SGI1-positive isolates
carry more or less identical
virulence gene repertoires.
limited to certain serotypes that are responsible for gastroenteritis.
Although a number of these studies give us an insight into the genetic
factors controlling Salmonella virulence behavior, many of these studies
are relatively low-throughput, as the genes chosen were identified based
on earlier findings. Recent studies, on the other hand, are exploring a
more comprehensive approach towards obtain a systems-level under-
standing of bacterial physiology by analyzing the expression of proteins
related to bacterial pathobiology under stress conditions that mimic
those experienced by bacteria during human colonization and infection
(Table 5). A major area of interest has been to identify the regulatory
systems that allow bacterial cells to identify and overcome host envi-
ronmental and immune signals. For example, Tran et al. (2016) con-
ducted a whole proteome analysis on wild-type and mutant S.
Typhimurium strains to identify proteins regulated by, and the overall
importance of, the RstA/RstB and PhoPQ two-component regulatory
systems (TCS) on bacterial pathogenesis, finding that while the PhoPQ
regulatory system was more extensive than the RstA/RstB system (14.9%
vs. 6.92%), the latter played a more significant role in regulating Sal-
monella motility and invasion [87]. This was similar to the results found
by Liu et al. (2015) [88]. On the other hand, Liu et al. (2017) and Karash
et al. (2017) performed a proteogenomic analysis of S. Typhimurium
under stress conditions (such as the production of reactive oxygen spe-
cies) initiated by macrophages, finding a significant downregulation in
histidine biosynthesis in the former, and upregulation in proteins
responsible for DNA repair, aromatic amino acid biosynthesis, Fe–S
cluster biosynthesis, iron homeostasis and H2O2 scavenging in the latter
study [89,90]. Li et al. (2019), on the other hand, focused on changes in
Salmonella metabolic pathways during host infection as indicators of its
survival and proliferation capabilities. The authors observed significant
upregulation of PhoPQ-regulated proteins, such as PgtE, PqaN, and
PhoN, and metal transporters such as SitAB, but downregulation of Sal-
monella flagellar proteins, such as fliC and fliFGLMNI, and chemotaxis
proteins such as Tsr, Tcp, Tar, and Trg, at 18 h post-infection of an in vitro
culture [91]. These, and additional findings by the authors indicate that
invasive Salmonella employ a non-motile lifestyle to support rapid pro-
liferation, and also display significant shifts in metabolism and respira-
tion cycles to optimize on the available conditions within the host,
similar to the results obtained by Liu et al. (2015) and Noster et al.
(2019). These studies provide a fairly detailed overview of the genotypic
and phenotypic controllers of bacterial physiology, which in turn can
help in fine-tuning the predictive models for bacterial dose-response [88,
92].
9
J. Chen et al.
Table 4
Comparative multiple virulence factor studies of Salmonella by PCR.
Authors, Description of the isolates
year,
country
1 Capuano 114 Salmonella strains of human
et al. (2013) origin (71/114) or isolated from
[22], Italy food (43/114) collected from
2010 to 2012, including only the
S. Enteritidis, S. Typhimurium,
S. 4 [5],12:i: .
2 Jaradat et al. 18 S. Enteriditis isolates
(2014) [77], obtained from raw poultry
North samples compared with 7
Jordan outbreak strains.
3 Campioni 128 S. Enteritidis strains isolated
et al. (2012) from human feces (67) and food
[114], Brazil (61) between 1986 and 2010 in
Brazil.
4 Rowlands 237 Salmonella strains isolated
et al. (2014) from food sources between 1983
[115], Brazil and 2007, among them 42 were
isolated from foods associated
with foodborne salmonellosis
and 195 from food samples not
associated with outbreaks or
sporadic cases.
5 Mohamed 309 S. Typhimurium and S.
et al. (2014) Kentucky isolates recovered
[78], U.S. from pre- and post-chill whole
broiler carcasses.
6 Diarra et al. 193 Salmonella isolates
(2014) [85], recovered from commercial
Canada farms
7 Graziani 112 strains of S. Napoli isolated
et al. (2011) in Italy from human cases, foods
[23], Italy of animal origin, and the
environment.
8 Saroj et al. 110 Salmonella isolates of 6
(2008) serovars from sprouts, fish, food
[116], India and blood, in India.
9 Majtan et al. 14 S. Typhimurium, 7 S.
(2007) [82], Kentucky and 3 S. Enteritidis
Slovakia from patients suffering from
various forms of
gastroenterocolitis.
Genes detected by PCR
PCR for mobile virulence
genes (gipA, gtgB, sopE, sspH1,
sspH2, sodC1, gtgE, spvC, pefA,
mig5, rck, srgA).
A phylogenic tree based on
16S rRNA sequencing was
constructed to cluster the S.
Enteritidis isolates.
ERIC-PCR and PFGE for
clustering.
PCR for SPI-1, SPI-2 and
virulence-associated plasmid
(13 genes: invA, sipA, sipD,
sopB, sopD, sopE2, ssaR, sifA,
spvB, prot6E, flgK, fljB flgL).
PCR for virulence genes of
spvC, invA, sefA and pefA.
Pulsed field gel
electrophoresis (PFGE) for
isolates comparison, and PCR
for three virulence factors
(InvA, pagC, spvC).
PFGE for isolates comparison
PCR for two virulence factors
(InvA, spvC)
PFGE for cluster analysis, PCR
for 10 virulence genes,
including five targets (avrA,
ssaQ, mgtC, spi_4D, and sopB)
located on the SPI 1–5, three
targets (gipA, sodC1, and
sopE1) on prophages, spvC on
plasmid, and one (bcfC) on a
fimbrial cluster.
PCR (SPI-8 and SPI-10).
Oligonucleotide microarray
and PCR confirmation (16
pathogenicity-related markers
including genes coding for
effector proteins associated
with SPIs and fimbriae).
10
Journal of Agriculture and Food Research 2 (2020) 100045
Phenotypic analysis Main findings of the study
59 different virulence profiles
were highlighted.
Isolates (1) The most virulent strains
representing each were isolated from poultry
cluster were further samples and had an LD50 of
studied for their 1.55  105 CFU.
virulence in normal (2) Some of the outbreak
adult Swiss mice. isolates were highly virulent
while others were avirulent,
which might be due to a
variation in susceptibility of the
mouse to different S. Enteritidis
isolates.
The strains isolated from
sporadic cases were
undistinguishable from the
outbreak strains.
The pathogenic potential is
highlighted with the presence
of the four virulence genes in S.
Enteritidis.
Production of Significant difference was
aerobactin and found in the production of
colicin. aerobactin and colicin between
S. Typhimurium and S.
Kentucky isolates (S.
Kentucky > S. Typhimurium).
10 distinct virulence profiles
(VP) were tested, but VP1-2-3
included 99 out of 112 strains.
These three profiles are similar
to each other, with single-gene
variations; compared to VP1,
VP2 lacks the bcfC gene,
whereas the VP3 lacks the
spi_4D gene.
SPI-8 and SPI-10 are serovar
specific, which is contradictory
to previous statement “SPI-8
and SPI-10 are widely
distributed among Salmonella
serovars”.
Virulence genes specifically
focused on
The genes showing the
highest homology were
those related to the
presence of prophages
(gipA, gtgB, sopE, sspH1,
sspH2, sodC1, gtgE), while
the genes related to the
presence of plasmids were
less frequently detected
(spvC, pefA, mig5, rck, srgA).
Almost all strains (97.6%)
harbored all 13 virulence
genes.
The invA was detected in all
strains; spvC and pefA were
found in 48.1% and 44.3%
of strains, respectively; sefA
was detected in 31.6% of
the strains and only among
S. Enteritidis.
Chilling has no effect on the
tested virulence factors.
The invA and spvC were
found in 97.9% and 25.9%
of the isolates, respectively,
with 33 (71.7%) of the 46 S.
Typhimurium isolates and
17 (65.4%) of the 26 S.
Enteritidis isolates carrying
both invA and spvC.
All the S. Napoli strains
lacked spvC, the avrA gene
in SPI, present in about
100% of the main serovars,
was detected in 5 out of
112 S. Napoli strains only.
Minimal variability of
virulence factors encoded
by SPIs, two S.
Typhimurium isolates were
defective in spaO and three
S. Kentucky strains were
(continued on next page)
J. Chen et al.
Table 4 (continued )
Authors, Description of the isolates Genes detected by PCR
year,
country
10 Chen et al. 12 Salmonella and 7 E. coli a PCR based microarray (10
(2005) isolates recovered from retail Salmonella virulence genes
[117], U.S. ground meat and food animals. including virulence plasmid,
SPI-1 and SPI-5, flagella).
11 Biswas et al. 67 Salmonella strains belonging PCR for bapA
(2011) to 34 serovars isolated from
[118], India diverse sources in India
including both reference strains
and field isolates from different
sources.
PFGE – Pulsed field gel electrophoresis.
MLST – Multi-locus sequence typing.
PCR – Polymerase chain reaction.
NTS – Non-typhoidal Salmonella.
SPI – Salmonella pathogenicity islands.
6. Status and potential of integration of WGS with QMRA for
salmonella
QMRA is a widely applied method that partially or wholly in-
corporates factors from the farm-to-consumption paradigm to identify
measures (or control steps) that can be taken to prevent and reduce mi-
crobial contamination along the (farm-to-fork) pathway. QMRA for Sal-
monella has been carried out worldwide in eggs and in common food
animals including meats such as broiler chicken, turkey, pork, and beef
[93–96], nuts [97], leafy greens [98], and more specifically, retail,
catering [99], and pet food [8]. QMRA methods, driven and challenged
by better data and the inclusion of uncertainty and variability, have
advanced greatly over the past two decades, allowing risk assessors to
describe the physical and biological processes of Salmonella across the
farm-to-fork pathway.
The nature of risk of Salmonella infection in humans and animals
depends on a variety of bacterial and host factors and their complex
interactive environment. This is a point that current risk assessment
strategies have not sufficiently addressed, despite the increase in focus on
the importance of the biological variations in Salmonella on predictive
microbiology [100]. The variations in pathogenicity and host adaptive-
ness of Salmonella might directly and immensely influence the
dose-response evaluation step, as well as other steps in risk assessment
[101,102]. As mentioned previously, Salmonella is classified into various
serovars. However, to date, published risk assessment of Salmonella has
not integrated serovar data or specified a certain serovar, although some
have mentioned that the difference in pathogenic potential presented as a
significant uncertainty factor [8,94–96,101,102].
Serovar is determined by the expression of a few surface antigens that,
in theory, can only provide limited information about the pathogenicity of
different Salmonella strains. Furthermore, classification by serovar may be
influenced by the relative occurrence of different serovars in different
geographic locations [10]. In other words, surface antigens are only in-
direct markers that tell us little about the pathogenic phenotype, providing
limited information for epidemiological investigations. The fact that ge-
netic differences within a serovar can have profound influence on the
pathogenicity of the isolate has already been noted; for example, S. Par-
atyphi B strains from systemic infections always lack the avrA gene but
contain sopE1, whereas those from enteric infections generally display the
opposite genetic pattern [103]. Compared to surface antigens, the geno-
type of an organism at dozens or hundreds of loci provides a greater po-
tential for revealing its human health risk [102,104].
11
Journal of Agriculture and Food Research 2 (2020) 100045
Phenotypic analysis Main findings of the study Virulence genes specifically
focused on
missing sptP located within
SPI-1.
Each Salmonella isolate
harbored virulence genes
including sipA, sipB, sipC,
and sop that reside on SPI-1,
SPI-5, as well as other
virulence factors such as
pagC, with variance in
virulence plasmid and invA.
BapA gene was found to be
highly conserved in Salmonella.
Moreover, the occurrence of frequent horizontal gene transfer among
pathogen strains could cause strain discrepancy, which could cause false-
negative PFGE results in the event of an actual transmission, which in
turn could contribute significantly to the acquisition of false-negative
results in Salmonella [46]. The first significant application of WGS that
led to source identification of a Salmonella foodborne outbreak was re-
ported in 2011 [16]. Epidemiological investigations into this multistate
outbreak (300 illnesses reported from 44 states and the District of
Columbia in the U.S.) of S. Montevideo which occurred between 2009
and 2010 (http://www.cdc.gov/salmonella/montevideo/) indicated
black and red peppers used to prepare salami as being the source of the
illness. Subsequent investigations into the supply chain of the spices
established the link between patients, the food product manufacturer,
and the ingredients supplier, which was confirmed by WGS of 35 strains
of Salmonella [16].
Given the evident genotypic plasticity and anticipated diversity of
virulence of Salmonella, the robust and high-resolution genomic data
provided by WGS has yielded new insights into the pathogenic trans-
mission pathways. These, in turn, have provided researchers with the
possibility of correlating disease manifestation with the diversity in
pathogenic genomic sequences within a geographic area or food category
to anticipate changes in pathogenicity, thereby facilitating research into
the behavior of Salmonella along the food chain. An example of this is
when Duchene et al. (2016) estimated the evolutionary rates of Salmo-
nella and other pathogenic species to understand the time-scale of genetic
diversification, which in turn would help in understanding major
evolutionary and ecological processes [105]. Ultimately, in 2018, re-
searchers from CFSAN presented a framework for combining the counts
of nucleotide differences (in terms of single nucleotide polymorphisms
(SNPs)) with evolutionary phylogenetic trees and bootstrap support to
interpret WGS data and analytical results, in order to determine if more
than one species of bacteria arose from the same source of contamination
[106].
Despite these advances in WGS and WGS-related bioinformatics
support, currently available Salmonella serovar data does not have suf-
ficient granularity for integration into risk assessment. Therefore, at this
point, grouping Salmonella according to genotypes attributable to human
health risk would be immensely more meaningful to public health than
species- or serovar-level classification and analyses, indicating that
genomic analyses will allow for better incorporation of variability among
bacterial strains in QMRA models [101,104]. To date, very few papers
have managed to directly integrate genomic data into health risk
J. Chen et al. Journal of Agriculture and Food Research 2 (2020) 100045

Table 5
Comprehensive proteomic or genoproteomic studies of Salmonella.
Authors, Description of the isolates Primary purpose of the study – Methods used Main findings of the study
year, microbial function to be elucidated
country

1 Tran et al. S. Typhimurium wild type strain Comparative analysis of the Proteins extracted from bacterial cell The RstA/RstB and PhoPQ systems
(2016) [87], ATCC14028, and ΔPhoPQ, functional networks under the control cultures grown under minimal were found to regulate 80 and 85
China & U.S. ΔrstB::Cm, and Δudp::Km mutants. of the PhoP/PhoQ and RstA/RstB conditions and subjected to liquid metabolic pathways, respectively.
two-component regulatory systems chromatography-tandem mass The PhoPQ system overrepresented
(TCS), which regulates pathogenesis. spectrophotometry (LC-MS/MS). 20 biological pathways, while the
RstA/RstB pathway overrepresented
only 6.
8 proteins were repressed by both
pathways, including 4 ribosomal
proteins (RpmB, RpsT, RpsO, and
RplY) and 2 primary metabolic
process-related proteins (FabZ and
TtcA).
RstA/RstB system was found to
specifically regulate the expression of
proteins responsible for pyrimidine
metabolism, iron acquisition,
Salmonella motility, and invasion.
2 Liu et al. S. Typhimurium SL1344 wild-type, Comprehensive proteomic expression Bacterial proteins isolated from Salmonella employs a borad range of
(2015) [88], ΔentC and ΔfeoAB mutants. of S. Typhimurium upon invasion and infected human epithelial (HeLa) cells adaptation mechanisms to survive in
China infection of nonphagocytic epithelial and subjected LC-MS/MS. host intracellular environments.
cells. Since Salmonella encounters severe
metal ion starvation in host epithelial
cells and upregulates iron,
manganese, and zinc-uptake systems
(EntC, IroN, IroC, CirA, ZnuA, ZnuC,
SitABCD, and FepA) at 6 h post-
infection.
Salmonella also upregulates the
PhoPQ regulatory system, and several
proteins from SPI-2 such as the SsaQn,
PipB, SseL, PgtE, and PagN, which are
important for its intracellular survival
and proliferation.
3 Liu et al. S. Typhimurium S344 wild type Comprehensive characterization of Bacterial proteins isolated from At 18 h post-infection, significant
(2017) [89], strain, and mutant ΔpfkAB, Δgnd, bacterial metabolism, motility, and infection HeLa cells and subjected to downregulation was observed in
China ΔpflB, ΔpurM, ΔdeoC strains. chemotaxis in host intracellular LC-MS/MS. Salmonella flagellar (FliC, FliF, FliG,
conditions. FliL, FliM, etc.) and chemotaxis
(CheA, CheW, CheZ, and CheY)
proteins, indicating loss of motility
and chemotaxis.
Late in the infection stages,
additionally, the tricarboxylic acid
(TCA) cycle and aerobic and
anaerobic respiration pathways were
repressed, leading to utilization of
glycolysis, pentose phosphate
pathway, mixed acid fermentation,
and nucleotide metabolism.
These results indicate the specific
mechanisms and factors responsible
for bacterial fitness during host
infection.
4 Karash et al. S. Typhimurium ATCC 14028s Determine Salmonella proteomic and Analysis conducted using electrospray 121 and 125 proteins were
(2017) [90], wild type strain, mutagenized with genotypic response to hydrogen ionization (ESI)-LC-MS/MS. upregulated and downregulated,
U.S. E. coli SM10 λpir. peroxide produced by macrophages respectively, under hydrogen
during invasion and infection. peroxide (oxidative) stress conditions.
Specifically, this study found that the
expression of amino acid biosynthesis
proteins aroB, aroD, aroE_2, aroK, and
aroA were significantly associated
with fitness under the stress
condition.
Additionally, proteins encoded by
ahpCF, tsaA, tpx, sodA, sodB, sodCI,
and sodCII played a significant role in
oxygen scavenging, and hence
promoting bacterial survival in within
the macrophage system.
5 Li et al. S. Typhimurium wild type strains Compare proteomic expression of S. Salmonella proteins isolated from Overall, many proteomic features
(2019) [91], SL1344 and SL14028s. Typhimurium from infected infected HeLa and human were shared between the two types of
China host cells, indicating a similarity in
(continued on next page)

12
J. Chen et al. Journal of Agriculture and Food Research 2 (2020) 100045

Table 5 (continued )
Authors, Description of the isolates Primary purpose of the study – Methods used Main findings of the study
year, microbial function to be elucidated
country

6 Noster et al. Wild type strain - S. Typhimurium
(2019) [92], NCTC12023, and isogenic mutant
Germany strains ΔssaV::FRT, defective in
SPI2-T3SS, and ΔsseF::FRT,
defective in the SPI2-T3SS effector
protein SseF.
macrophages against those from
infected epithelial cells (HeLa).
Compare the intracellular proteomic
response of wild-type S. Typhimurium
to mutant strains with aberrant
Salmonella-induced filament (SIF)
architecture against nutritional stress
and host cell defense mechanism.
macrophage (RAW 264.7) cell lines
analyzed by LC-MS/MS.
Salmonella proteins isolated from
infected macrophage culture and
subjected to liquid chromatography-
mass spectrophotometry.
intracellular environmental cues. This
included the induction of metal ion
transportation and utilization
pathways and repression of TCA and
anaerobic respiration pathways.
However, the time required for
intracellular adaptation varied,
especially in the cases of SPI-1 T3SS
and SPI-2.
Uniquely, Salmonella expressed
protein signatures indicative of
histidine biosynthesis when infecting
macrophage cultures, indicating a
path for Salmonella suppression in this
host cell type.
Infection-associated proteins such as
SlyA/B, PhoP/N, Fur, Hfq, YdcR,
SodC1 and PntA, were upregulated in
the ΔsseF mutant, allowing for
increased intracellular replication
under macrophage stress conditions.
Induction of SIFs required for
sufficient nutritional supply.
While nutritional limitations slightly
reduces ΔsseF mutant replication, the
mutant ΔssaV displays the additional
inability to neutralize host defense
mechanisms.

SPI – Salmonella pathogenicity island.

T3SS – Type III secretion system.
LC-MS/MS – Liquid chromatography – tandem mass spectrophotometry.
TCA – tricarboxylic acid cycle.

assessment [60,107–109]. Previously, Pielaat et al. (2015) explored the
integration of WGS data in hazard characterization of Shiga
toxin-producing E. coli (STEC) O157 [60] by conducting a Genome Wide
Association Study (GWAS), wherein genetic data (virulence SNPs) was
correlated with phenotypic data (in vitro adherence to epithelial cells as a
proxy for virulence). On the other hand, Gale et al. built a dose–response
model for RNA viruses by breaking down initial infection in the gastro-
intestinal tract into the component steps at the molecular level and
assigning probabilities to each step, which led to the conclusion that the
molecular mechanisms are not sufficiently well understood at present to
enable quantitative estimation of probabilities on the basis of omics data
[107]. More recently, Njage and colleagues employed machine learning
approaches to link genome composition with clinical outcome in
humans, by identifying virulence genes that were important predictors of
higher frequency of illness for Listeria monocytogenes and STEC to update
the hazard characterization paradigm of the risk assessment framework
[108,109]. However, the potential of such an estimation must not be
neglected. All of these studies took a baby step towards the application of
genomic data of E. coli, Listeria and RNA virus (whose genomic content
and phenotypic traits are simpler than those of Salmonella) in a proba-
bilistic framework. In the case of Salmonella, high levels of genome
similarity do not necessarily imply similar behavior in the food chain or
similar levels of pathogenic phenotype, since small genetic changes may
result in large phenotypic differences [39,110], which increases the
complexity of the data integration process.
The fields of QMRA and genomic analyses have both seen significant
development over the past 15 years [60,101,104,108–110]. Genomic
approaches offer the potential to both inform QMRA and transform how
it is conducted. QMRA of Salmonella if further challenged by the
increasingly comprehensive descriptions of the complex, dynamic
behavior of bacteria within the context of the host provided by WGS data.
Therefore, there is a long way to go before WGS data can be routinely
used in QMRA of Salmonella. It should also be noted that the use of WGS
data alone to predict pathogenicity could give rise to further uncertainty
as the existence of a particular virulence or antibiotic resistance gene is
not necessarily indicative of phenotypic expression. Phenotypic hetero-
geneity may also be caused by mutations and acquisition of genes
through horizontal gene transfer, as well as by epigenetic mechanisms
that are not accompanied by changes in the genome content [101].
It can be safely summarized that the challenge of converting primary
WGS data into useful public health risk assessment remains unmet, and
that experience with such data is quite limited at this point. In addition,
there is no doubt that gene expression studies, transcriptomics, prote-
omics, metabolomics, high-throughput phenotyping approaches, and
other “omics” studies, coupled with genomic studies, would be more
informative in the future QMRA of Salmonella.
7. Future recommendations
Based on the above findings, it is safe to conclude that multiple in-
dividual virulence genes are variably distributed in Salmonella. It is quite
unrealistic to expect a single locus to be responsible for all pathogenic
traits; instead, a complicated combination of genes is more likely to
contribute to the overall virulence phenotype. It can be concluded that
comparative studies analyzing the differences in genome content among
different “groups” of Salmonella must be performed to assess the impor-
tance of differences in virulence characteristics among strains. A more
realistic approach to relate phenotypic differences with genetic de-
terminants would be to compare closely related bacterial lineages that
exhibit distinct biological properties. In this way, genes that are directly
associated with special pathogenic phenotypes could be maximally
focused. Furthermore, a majority of the studies continue to focus on the
commonly detected gastroenteritis serovars (viz. S. Typhimurium and S.
Enteriditis); therefore, future research should include more serovars.
Due to the lack of both standard sequencing and post-bioinformatics
methods for WGS and corresponding phenotypic analysis, researchers are

13
J. Chen et al.
currently employing different platforms and bioinformatics tools for
WGS analysis (and for the analysis of its metadata). Metadata that would
be required to perform such comparative research would include viru-
lence gene profiling, phylogenetic analyses, phenotypic characteristics,
and the resulting epidemiological traits. The main limiting factor, how-
ever, would not be the cost of sequencing; rather, it would be how to
assemble, process, and handle large amounts of WGS data (as well as its
metadata) in a standardized way that will make the information useful, in
this case for food safety risk assessment. Therefore, further research must
be directed at the following focal points:
 Metadata that could be of value in determining the role of food in the
epidemiology of isolates is rare and, when present, incomplete.
Detailed epidemiological information corresponding to molecular
data should be collected and provided in the future.
 Genomics can be used to identify genes responsible for a variety of
factors. Experimental data remains essential for improving the un-
derstanding of health risk posed by foodborne pathogens.
 Methods to directly screen for virulence-associated phenotypes in
animal models are required to take a more comprehensive approach
to define genes essential for in vivo infection.
 It is unrealistic and meaningless to attempt the mapping of microbial
WGS data consisting of thousands of loci onto a probabilistic scale for
risk. Considering the genotype-phenotype diversity, another potential
way is to model the variability of multiple virulence genes identified
by WGS among the various Salmonella isolates within one food
category or one serovar. Quantification and calibration of the dif-
ferences (such as SNP variants) between genomes will facilitate the
categorization of Salmonella, allowing us to treat them accordingly.
 Genome Wide Association Studies (GWAS), which have been devel-
oped in other areas (but not in foodborne microorganisms), could
facilitate the management of large sets of phenotype and genotype
data, analysis of phenotypic variation and heritability, and most
importantly, generation of a refined set of candidate single nucleotide
polymorphisms (SNPs).
 The main biological process or (potentially) food processing chain can
be broken down into its component steps such as (surviving the
various) process conditions and time required to initiate growth or
invasion of gut epithelium, which would guide the data requirements
(e.g. specific gene expression data and phenotypic data as cells grow).
 Recent outbreaks in the U.S. have been linked to vegetables, nuts, and
fruits, in addition to traditional sources (food animals). Therefore,
Salmonella strains could have significant phenotype diversity and
potential genomic variability in host specificity or colonization that
could be worth looking into. The potential findings might contribute
significantly to the risk assessment of these food sources.
8. Conclusion
It should be mentioned that the primary aim of this review was to
pool genomic data with its corresponding phenotypic data in order to
potentially conduct a quantitative modeling study to correlate genotypes
with their corresponding phenotypes. However, as our literature search
has identified gaps and inconsistencies in this research such as the
limited number of related studies, diversity in the methodology of
phenotypic testing, and variability in data obtained from even the most
common epithelial cell invasion assays, conducting a quantitative anal-
ysis at this point might not be feasible. Although comparative genomic
studies for Salmonella using WGS has seen a considerable amount of in-
terest over the past decade, a larger number of studies have reported the
use of comparative genomic hybridization (CGH) and PCR-based tech-
niques for comparative genomic analysis of Salmonella. The reviewed
studies that attempted to correlate phenotypic differences in different
serovars with genetic variations in order to gain insights into the effect of
genome variations on apparent differences in prevalence and other fea-
tures provide limited information. Additionally, the transition to
14
Journal of Agriculture and Food Research 2 (2020) 100045
genomic technologies in food safety risk assessment is currently in flux
without a clearly defined path and methodology. However, with the
accumulation and standardization of WGS data, the normalization and
availability of advanced proteomics, and increasing utilization of ma-
chine learning and advanced data analytic techniques, it would be
possible to make the best use of all available data in order to improve the
efficiency of QMRA in the near future. This will undoubtedly provide
new insights and inform actions to mitigate the risk caused by Salmonella
in chicken, as well as other pathogenic agents in food.
Declaration of competing interest
None.
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