frontiers 

in Microbiology 

OPEN ACCESS 

ORIGINAL RESEARCH 

published: 19 November 2021 

doi: 10.3389/fmicb.2021.777817 

Edited by:

Nilton Lincopan, 

University of São Paulo, Brazil 

Reviewed by:

Herrison Fontana, 

University of São Paulo, Brazil 

Jing Han, 

National Center for Toxicological 

Research (FDA), United States 

*Correspondence:

Shaohua Zhao 

shaohua.zhao@fda.hhs.gov 

†These authors have contributed 

equally to this work 

Specialty section:

This article was submitted to 

Antimicrobials, Resistance 

and Chemotherapy, 

a section of the journal 

Frontiers in Microbiology 

Received: 15 September 2021 

Accepted: 19 October 2021 

Published: 19 November 2021 

Citation:

Li C, Tyson GH, Hsu C-H, 

Harrison L, Strain E, Tran T-T, 

Tillman GE, Dessai U, McDermott PF 

and Zhao S (2021) Long-Read 

Sequencing Reveals Evolution 

and Acquisition of Antimicrobial 

Resistance and Virulence Genes 

in Salmonella enterica. 

Front. Microbiol. 12:777817. 

doi: 10.3389/fmicb.2021.777817 

Long-Read Sequencing Reveals 

Evolution and Acquisition of 

Antimicrobial Resistance and 

Virulence Genes in Salmonella

enterica

Cong Li1†, Gregory H. Tyson1†, Chih-Hao Hsu1, Lucas Harrison1, Errol Strain1,

Thu-Thuy Tran1, Glenn E. Tillman2, Uday Dessai3, Patrick F. McDermott1 and

Shaohua Zhao1

*

1 Center for Veterinary Medicine, United States Food and Drug Administration, Laurel, MD, United States, 2 Food Safety 

and Inspection Service, United States Department of Agriculture, Athens, GA, United States, 3 Food Safety and Inspection 

Service, United States Department of Agriculture, Washington, DC, United States 

Salmonella enterica is a signifcant and phylogenetically diverse zoonotic pathogen. To 

understand its genomic heterogeneity and antimicrobial resistance, we performed longread sequencing on Salmonella isolated from retail meats and food animals. A collection 

of 134 multidrug-resistant isolates belonging to 33 serotypes were subjected to 

PacBio sequencing. One major locus of diversity among these isolates was the 

presence and orientation of Salmonella pathogenic islands (SPI), which varied across 

different serotypes but were largely conserved within individual serotypes. We also 

identifed insertion of an IncQ resistance plasmid into the chromosome of fourteen 

strains of serotype I 4,[5],12:i:– and the Salmonella genomic island 1 (SGI-1) in fve 

serotypes. The presence of various SPIs, SGI-1 and integrated plasmids contributed 

signifcantly to the genomic variability and resulted in chromosomal resistance in 

55.2% (74/134) of the study isolates. A total of 93.3% (125/134) of isolates carried 

at least one plasmid, with isolates carrying up to seven plasmids. We closed 233 

plasmid sequences of thirteen replicon types, along with twelve hybrid plasmids. Some 

associations between Salmonella isolate source, serotype, and plasmid type were 

seen. For instance, IncX plasmids were more common in serotype Kentucky from 

retail chicken. Plasmids IncC and IncHI had on average more than fve antimicrobial 

resistance genes, whereas in IncX, it was less than one per plasmid. Overall, 60% 

of multidrug resistance (MDR) strains that carried >3 AMR genes also carried >3 

heavy metal resistance genes, raising the possibility of co-selection of antimicrobial 

resistance in the presence of heavy metals. We also found nine isolates representing 

four serotypes that carried virulence plasmids with the spv operon. Together, these 

data demonstrate the power of long-read sequencing to reveal genomic arrangements 

and integrated plasmids with a high level of resolution for tracking and comparing 

Frontiers in Microbiology | www.frontiersin.org 1 November 2021 | Volume 12 | Article 777817 
Li et al. Genomic Diversity of MDR Salmonella enterica 

resistant strains from different sources. Additionally, the fndings from this study will help 

expand the reference set of closed Salmonella genomes that can be used to improve 

genome assembly from short-read data commonly used in One Health antimicrobial 

resistance surveillance. 

Keywords: Salmonella, multidrug resistance (MDR), plasmid, Salmonella genomic island (SGI), Salmonella

pathogenicity island (SPI) 

INTRODUCTION 

Salmonella enterica is an important zoonotic pathogen that 

causes over one million illnesses in the United States each 

year (Scallan et al., 2011). S. enterica are classically subdivided 

into serotypes and over 2,600 serotypes have been identifed 

thus far. While many serotypes may be capable of causing 

infections in humans and animals, a limited number of 

serotypes cause most human infections in the United States. 

Recent advancements in whole genome sequencing (WGS) oer 

a unique opportunity to dissect and investigate Salmonella 

serotypes at the nucleotide level and to further our understanding 

about notable evolutionary changes. The main features associated 

with S. enterica evolution include acquisition and recombination 

of mobile genetic elements such as genomic islands, transposons, 

integrons, and plasmids, among others (Partridge et al., 2018). 

An in-depth analysis of these features will help us to understand 

drivers of resistance, host and environmental adaptations, and 

sources of resistant Salmonella infections. 

While most Salmonella infections are self-limiting, serious 

infections can require antimicrobial therapy (Tack et al., 

2020). Antimicrobial resistance (AMR) can compromise therapy, 

increase healthcare costs, and cost lives (Abebe et al., 2020). In 

Salmonella, AMR is typically attained by horizontal acquisition 

of antimicrobial resistance genes (ARGs), although chromosomal 

mutations also play a role (McDermott et al., 2016). 

One way that Salmonella strains acquire ARGs is through 

acquisition of plasmids (Emond-Rheault et al., 2020). Plasmids 

carry not only ARGs, but also heavy metal and disinfectant 

resistance genes, which may contribute to co-selection for 

AMR (Vijayakumar and Sandle, 2019). The types of plasmids 

that Salmonella carry can vary considerably, as they may 

include species-specifc non-conjugative plasmids, or conjugative 

plasmids found widely among Enterobacterales (Redondo-Salvo 

et al., 2020). Some plasmid types are highly associated with 

specifc serotypes and sources (Zhao et al., 2020), thus plasmids 

provide important information for outbreak investigations and 

AMR source attribution. Traditionally, incompatibility plasmid 

types have been used to assign plasmids into dierent groups 

based on plasmid replication machinery (Carattoli, 2013). This 

approach does not account for all plasmid types and it is often 

unclear which replication machinery is dominant, especially in 

hybrid plasmids arising from recombination (Hsu et al., 2019). 

Characterization of plasmids and other resistance elements 

in Salmonella has been studied extensively by WGS. The use 

of short-read sequencing in conjunction with programs such as 

PlasmidSpades, PLACnet, or others have helped expand analyses 

of genomes derived from short-read sequencing data (de Toro 

et al., 2014; Antipov et al., 2016). There have been relatively few 

large-scale, long-read sequencing studies, which can yield more 

complete genomic information with higher resolution. 

Aside from plasmids, ARGs also are commonly carried by 

chromosomally encoded Salmonella genomic islands (SGIs). 

SGI-1 was frst reported in S. Typhimurium DT104 in 2001. It 

contained a 27 kb backbone plus a 15 kb complex with a class 1 

integron, with ARGs conferring resistance to fve antimicrobial 

classes (Boyd et al., 2000). Dierent variants of SGI-1 have been 

described, with a diversity of ARG alleles in multidrug resistance 

(MDR) regions (Hall, 2010). Additional SGIs, including SGI-0, 

SGI-2, SGI-3, and SGI-4, have been identifed based on genomic 

structure and resistance gene contents. Both SGI-0 and SGI-2 are 

in the same location as SGI-1 and shared the SGI-1 backbone 

sequence (Levings et al., 2008; de Curraize et al., 2020). SGI-3 and 

SGI-4 were initially described as distinct SGIs, but they are in the 

same chromosomal location, have the same sequence backbone 

structure and are considered the same SGI (Arai et al., 2019; 

Branchu et al., 2019). SGI-4 did not carry AMR genes, instead 

it carried 24 heavy metal resistant genes (HMRGs) (Arai et al., 

2019). Together the acquisition of AMR determinants, mobile 

genetic elements contribute to the genomic diversity found in 

Salmonella. 

Salmonella pathogenic islands (SPIs) play a pivotal role in 

Salmonella virulence (Hensel, 2004; Rychlik et al., 2009). There 

are 24 known SPIs and several are associated with particular 

mechanisms of virulence (Cheng et al., 2019). SPI-1 and SPI-2, 

which encode type III secretion systems, are conserved across 

Salmonella, along with SPIs 3-6, SPI-9, and SPI-11 (Jennings 

et al., 2017; Lou et al., 2019; Zhao et al., 2020). Most other SPIs 

are variable across serotypes and may account for dierences in 

virulence among Salmonella serotypes (Zhao et al., 2020). Thus, 

the presence and organization of these SPIs are important to 

understanding the evolution and pathogenicity in Salmonella. 

The history of Salmonella epidemiology has relied on various 

features to categorize strains. Following biochemical profling, 

serotyping has long been the basis of Salmonella strain typing and 

tracking. Later, plasmid profling by electrophoresis and pulsed 

feld gel electrophoresis (PFGE) were used. For AMR monitoring 

in national programs such as the United States National 

Antimicrobial Resistance Monitoring System (NARMS), 

minimum inhibitory concentration (MIC) testing followed by 

multiplex PCR and conjugation assays have been commonly used 

to track phenotypic and genotypic resistance. With the advent 

of aordable WGS, surveillance of AMR Salmonella and other 

pathogens can now be done routinely using short read DNA 

sequencing chemistries. While this provides a comprehensive 

picture of strain relatedness and gene carriage, closed genomes 

Frontiers in Microbiology | www.frontiersin.org 2 November 2021 | Volume 12 | Article 777817 
Li et al. Genomic Diversity of MDR Salmonella enterica 

are needed to reveal the detailed gene arrangements and 

structural changes. In this report, we describe the use of PacBio 

long-read sequencing to characterize 134 isolates, representing 33 

Salmonella serotypes, isolated from raw meats and food animals. 

This study helped us to elucidate the genomic structure and 

location of virulence and resistance genes, their colocation on 

mobile DNA elements, and how these traits relate to Salmonella 

evolution. We also proposed a new approach to simplify naming 

of SGIs based on their genomic position. 

MATERIALS AND METHODS 

Isolate Sources and PacBio Sequencing 

One hundred thirty-four isolates, representing 33 serotypes, 

were collected as part of routine surveillance by the NARMS. 

The sources of these isolates were chicken, turkey, beef, 

and pork products as well as cecal/gut samples collected 

at slaughter from swine, turkey, cattle, and chicken from 

2016–2018 across 31 dierent states. Isolates were selected 

for Pacifc Biosciences (PacBio) long-read sequencing to 

represent diverse resistance patterns including three pansusceptible isolates, diverse serotypes and dierent NARMS 

sources (Supplementary Table 1). 

For long-read sequencing, DNA libraries were prepared 

using a 10 kb template preparation protocol with SMRTbell 

template prep kit v 1.0. Sequencing was performed using Pacifc 

Biosciences technology on the Sequel platform with sequencing 

kit 3.0, as described previously (Tate et al., 2021). 

Sequencing data are available in BioProject 

PRJNA292661. Isolate-level accession numbers are listed in 

Supplementary Table 1. 

Resistance Gene and Plasmid 

Identifcation 

Antimicrobial resistance genes, biocide resistance genes, and 

HMRGs were identifed with AMRFinder Plus version 3.8 

(Feldgarden et al., 2019). The AMRFinder Plus virulence genes 

and ARGs outside the AMRFinder core genes were not reported, 

due to their limited relevance to this Salmonella study. 

To identify plasmid replicon sequences, we used 

PlasmidFinder with cutos of 90% identity and 60% length 

(Carattoli et al., 2014). The sequence of the spvRABCD operon 

was extracted from the plasmid pOU1115 carried by a S. Dublin 

strain (Accession DQ115388). A local blastn analysis with the 

same cutos was performed to identify the presence of this 

spv operon. 

Integrated plasmids were also identifed by a similar approach, 

with PlasmidFinder being used to identify replicons. In some 

cases, blastn analysis was conducted to identify similarity with 

known plasmids (Table 2). 

Salmonella Pathogenic Islands and 

Salmonella Genomic Island Identifcation 

Sequences of 24 SPIs were downloaded from GenBank to a local 

database (Fookes et al., 2011; Hayward et al., 2014; Cheng et al., 

2019; Hsu et al., 2019). The size of the SPIs ranged from 1.7 

to 133.3 kb, encoding 1–21 virulence genes. Due to the variable 

length and gene content of SPIs, specifc SPIs were identifed as 

present if any of its associated virulence genes in the database 

developed previously (Blondel et al., 2009; Suez et al., 2013; Zhao 

et al., 2020) were identifed with 85% identity and at least 70% 

length by blastn v.2.7 and anchored by blasting chromosome 

sequences with the reference SPIs sequence using 85% identity 

and at least 10% length. SPI-8 and SPI-13 have the same genomic 

location adjacent to tRNA-pheV (Espinoza et al., 2017), and 

seven isolates from four serovars carried 17 kb (70%) SPI-13 

sequence but without its virulence genes were given the name of 

SPI-13∗ in this paper. 

SGI-1 and potential variant sequences were initially identifed 

by blast with 85% identity and 70% length to 47 kb of 

reference SGI-1 sequence from S. Typhimurium DT104 

(Boyd et al., 2000). Further analysis to identify additional 

SGI-1 sequences involved identifying insertions between 

yidY(50

) and thdF(30), with seven additional SGI variants 

identifed. The existence of SGI-4 was discovered using a blast 

query of chromosomal sequences against SGI-4 reference 

(MN730129.1) with 85% coverage and 70% homology cutos. 

The additional SGI-4 was discovered by aligning the N16S319 

and other S. Alachua without resistance genes. The other 

islands with ARGs and/or HMRGs were discovered using 

comparisons to chromosomes within the same serotypes in 

isolates lacking ARGs and HMRGs. 

Phylogenetic Tree 

The program KSNP3.0 was used to generate Single Nucleotide 

Polymorphisms (SNPs) from a subset of 44 complete 

chromosomes to represent all 33 serotypes and cases of 

chromosomal heterogeneity within serotypes (Gardner et al., 

2015). Prior to SNP generation, the kmer size was chosen 

by Kchooser included in KSNP3.0 (Gardner et al., 2015). 

The maximum likelihood phylogeny tree was constructed 

by MEGA7.0 with 250 bootstraps. The clade including S. 

Montevideo and S. Schwarzengrund was placed at the root based 

on established literature (Worley et al., 2018). 

RESULTS 

Presence of Salmonella Pathogenic 

Islands and Arrangement in the 

Chromosome 

Assembly of long-read sequences produced circular closed 

chromosomes in 116 of the 134 Salmonella isolates, ranging in 

size from 4,492,868 bp in S. Bredeney to 5,073,615 bp in S. I 

4,[5],12:i:-. Nine isolates showed genomic size >5,000,000 bp, 

with six of them I 4,[5],12:i:-, two S. Agona and one S. 

Typhimurium (Supplementary Table 1). Our data showed 

that additional accessary genomic elements, such as phage, 

integration of plasmids in chromosome or SGI contributed to the 

larger genomes. 

Frontiers in Microbiology | www.frontiersin.org 3 November 2021 | Volume 12 | Article 777817 
A 

IOJ Fl8S004 Typhlmurlum 

IOJ Nl6S070 Typhlmurlum 

IOJ Fl85010 4,(S),12~:-

Fl85012 Typhlmuriu.m 

1111 Nl850993 14,(SJ,= :· 

N18S0175 S..lntpaul 

Fl85033 H•ldelberc 

'"' Nl 7S0166 14,(51,lZ;r:~ 

'"' F185006 Mu•nchen 

Nl 75129 Muenchen 

13 Fl8S049 Hadar 

'"' Nl75166 Hadar 

Fl85038 AnJ1tUn1 

Fl8S024 Newport 

Nl75922 Utchfleld 

Berta 

Fl85037 Dublin 

Nl7Sl ll E:ntuitldl1 

Nl 75192 Enterttldl1 ,. ,--------- Fl8S0 19 Ucanda ,. ._ _______ _ Nl 7S367 London 

'15 ._ ________ _, F18S048 lnfantls 

, .. 11D N18S1988 lnfanti1 

._ _________ F18S036 Ohio 

---------- F18S016 C.r-ro 

._ _________ N18S0824 IClatnbu 

100 N17S1441 Derby 

,----------"'"'"-4 N18S0357 Duby 

F18S029 Derby 

, -------- N16S319 Abchu■ 

._ ________ N18S0935 Kentucky 

N18Sl350 Albany 

F 185046 A.con a 

._ ________ Nl?S1545 kentudcy 

F18S018 Meluc ridls 

._ _______ -I N17S834 S.nhenberc 

100 N18S0991 S.nftenberc 

------- F18S023 oh nnuburc 

,------- F18S011 Montevideo 

----Fl8S015 lrandenburc 

.__ ___ Fl8S042 RHdlnc 

------- F18S001 Muenrter 

N17S1304 Schw r.r.•n1rund 

l ffl Nl&S0406 8redeney 

C 

Li et al. Genomic Diversity of MDR Salmonella enterica 

FIGURE 1 | Presence of SPIs in different Salmonella serotypes. (A) A phylogenetic tree was constructed based on chromosomal SNPs of each of the isolates. 

(B) SPI arrangements are listed with representative sequences of each serotype. Red indicates that SPIs are conserved in all serotypes, yellow indicates insertions of 

SGIs or plasmids. Other colors represent SPIs whose presence are variable among different serotypes/isolates. (C) The conserved structure across all serotypes is 

shown. 

Salmonella pathogenic islands contain a variety of genes 

that contribute to Salmonella virulence as part of Salmonella 

serotype evolution (Marcus et al., 2000). To assess the 

complement of SPIs that contribute to the diversity of 

chromosomal sequences from dierent serotypes, we constructed 

a phylogenetic tree using SNPs across 44 chromosomes from 

34 serotypes (Figure 1A). This tree refects the phylogenetic 

relationships among dierent Salmonella serotypes and is 

refective of their entire genomic content. 

The SPIs 1-6, SPI-9, SPI-11, and SPI-24 were present in all 

isolates (Figure 1B and Supplementary Table 1). SPI-24 was 

previously called a CS54 (Sabbagh et al., 2010), but later renamed 

as SPI-24 (Cheng et al., 2019). Each of these conserved SPIs 

were largely in conserved locations as well, except for large 

inversions encompassing multiple SPIs in serotypes Infantis, 

Muenchen, Typhimurium, and Enteritidis (Figures 1B,C). Three 

SPIs, including SPI-20, SPI-21, and SPI-22, were not identifed in 

our isolate collection. 

Frontiers in Microbiology | www.frontiersin.org 4 November 2021 | Volume 12 | Article 777817 
Li et al. Genomic Diversity of MDR Salmonella enterica 

Salmonella pathogenic islands were largely conserved within 

serotypes but showed varying degrees of diversity between 

serotypes, consistent with previous reports (Hsu et al., 2019; Zhao 

et al., 2020). As expected from the close relationship between S. 

Typhimurium and its monophasic variant S. I 4,[5],12:i:- (Ido 

et al., 2014), their complement of SPIs was identical (Figure 1). 

In fact, the large clade of related serotypes from Typhimurium 

to Litchfeld all had almost identical combinations of SPIs 

(Figure 1B). Only a few serotypes displayed SPI variability among 

their strains. Interestingly, none of the SPIs were serotypespecifc, as each of the 21 identifed SPIs were distributed among 

multiple serotypes. Large inversions were observed within some 

serotypes. For example, in one of two S. Enteritidis strains, a 

large fragment from SPI-6 to SPI-17 was inverted, and in one of 

the S. Infantis strains the region between SPI-12 to SPI-14 was 

inverted (Figure 1B). 

Acquisition of Genomic Islands and 

Associated Antimicrobial Resistance 

Genes and Heavy Metal Resistant Genes 

In this study, SGI-1 and SGI-1 variants were found among 12 

strains including serotypes Typhimurium (n = 5), Senftenberg 

(n = 2), Derby (n = 3), Saintpaul (n = 1), and Alachua (n = 1) 

(Table 1). The location of insertion of SGI-1 was consistent 

across these dierent serotypes, present between SPIs 4 and 3 in 

the assembled chromosomes. The backbone and ARGs in SGIs 

varied greatly in dierent strains. All fve S. Typhimurium SGI-1 

carried similar backbone structure and ARGs, aadA2-qacEdeltasul1-foR-tet(G)-blaCARB-2-qacEdelta as previously reported 

(GenBank accession number AF261825), except that in N17S016, 

whose SGI was 9 kb shorter and contained only sul1 and 

blaCARB−2 ARGs. 

The three S. Derby strains carried similar SGI-1 backbone 

structure (43.9–44.9 kb) as the S. Typhimurium strain, and all 

three carried a similar resistance gene cassette (Table 1), with 2 

to 4 ARGs and 4 mercury resistance genes. 

The remaining four SGI-1 sequences varied greatly in size, 

from 29.7 kb in a S. Saintpaul strain to 117.9 kb in a S. Senftenberg 

strain (Table 1). The structures and resistance gene content 

varied considerably, with 5–6 ARGs and 0–6 mercury resistance 

genes (Table 1). Interestingly, although the top NCBI blast hits 

were for Salmonella, all SGI-1s are found to be homologous 

or partially homologous (Table 1) to the genomic islands on 

Proteus. The S. Senftenberg SGI-1 was particularly notable since 

it appeared to have a hybrid origin with 51 kb aligning with 

other SGI-1 sequences and a 67 kb homology with that of 

Citrobacter (Table 1). 

SGI-4 (MN730129.1) was found in ten S. I 4,[5],12:i:- strains 

and was located between SPI-4 and SPI-6 (Table 1). As previously 

reported, it did not carry ARGs and only carried HMRGs pco, 

sil, and ars encoding for resistance to copper, silver, and arsenic, 

respectively (Bearson et al., 2020). 

An additional potential estimated 84 kb genomic island 

(Table 1) was found in S. Alachua (Table 1). It has some 

homology to SGI-4 with 95% identity and 40% length. Although 

not experimentally validated as an SGI, it has many similarities 

to SGI-4 including its location between SPI-4 and SPI-6 and 

presence of the sil and pco HMRGs. This region also has genes 

related to conjugative transfer and partitioning, indicating that it 

is likely a mobile element. 

Integration of Plasmids Into the 

Chromosome and Associated 

Antimicrobial Resistance Genes and 

Heavy Metal Resistant Genes 

IncQ plasmid replicons were detected in chromosomal sequences 

of fourteen S. I 4,[5],12:i:- isolates (Table 2). Interestingly, the 

location of IncQ plasmids was in the same region as the fjB 

gene encoding the phase two H antigen in S. Typhimurium, 

and the fjB region missing in S. I 4,[5],12:i:- (Figure 2). 

This fnding suggests a plasmid to chromosome recombination 

event that transformed strains from serotype Typhimurium 

to I 4,[5],12:i:-. In each of the S. I 4,[5],12:i:- genomes in 

this study the recombination event had introduced sul2 and 

in all but one isolate tetB, blaTEM−1 and aph(300)-Ib/aph(6)-Id 

were introduced. 

Another similar recombination event was detected in strain 

N17S166 of serotype I.4,[5],12:r:-. In this isolate a fragment 

of 152 kb from a IncHI plasmid (pN17S1352-1) carried mcr9.1, aadA1, aac(3)-VIa, sul1 and two HMRGs, pcoS and 

arsC, integrated into the chromosome between SPI-1 and SPI9 in the same region where S. Heidelberg carried a fjB 

gene (Table 2). 

There are additional examples of ARGs or HMRGs in the 

chromosome that may have resulted from plasmid integrations 

(Table 2). For three S. Typhimurium strains from retail 

chicken (N18S0597, N18S1595, and N18S2170), there are two 

chromosomal regions with ARGs and HMRGs. The frst is 

an up to 107 kb region located between SPI-6 and SPI16 with merR, tet(A), and sul2, and can be traced back 

to an IncC plasmid pN18S1634-2. The second region is 

about 85 kb and has over 70% alignment to a previously 

published IncHI plasmid (nucleotide accession MH287085.1). 

This 85 kb region carries fve ARGs and 16 HMRGs. Another 

common insertion included a 31.6 kb element with silver 

and copper resistance genes. This insertion was found in the 

chromosomes of nine isolates, including serotypes Muenster, 

Johannesburg, Senftenberg, Heidelberg, Schwarzengrund, and 

Agona, and across multiple animal sources (Table 2). This 

insertion has high sequence homology with the IncHI plasmid 

pF18S044-1 (Table 2). These fndings reveal how plasmids or 

their remnants can contribute to the chromosomal acquisition 

of ARGs and HMRGs. 

The most common serotypes with chromosomal ARGs were 

Typhimurium (20/24 isolates), I 4,[5],12:i:- (15/16), Agona 

(10/10), and Heidelberg (7/7). In contrast, serotypes Kentucky 

(0/11) and Reading (0/6) did not have any chromosomal ARGs. 

Among the nine isolates that did not carry any plasmids, no 

chromosomal ARGs were found in three isolates (N17S192, 

N17S312, and N18S1429) which were pan-susceptible to all 

antimicrobials tested, fosA was found in two (N18S0476 

and N18S0722), and the remaining four (N17S107, N17S834, 

Frontiers in Microbiology | www.frontiersin.org 5 November 2021 | Volume 12 | Article 777817 
TABLE 1 | Putative Salmonella genomic islands in the genomes of Salmonella isolates. 

Frontiers in Microbiology | www.frontiersin.org 6 November 2021 | Volume 12 | Article 777817 Strain ID Serotype SGI Size Accession Position Top non-Salmonella hit Accession# 

of organism and coverage, identity F18S012 Typhimurium SGI-1 47,722 OK209931 SPI-3, SPI-4 Proteus mirabilis (89%, KJ186152.1 

F18S028 100%) 

F18S031 N16S132 N17S016 Typhimurium SGI-1 38,450 OK209935 SPI-3, SPI-4 Proteus mirabilis (89%, KJ186152.1 

100%) N17S1441 Derby SGI-1 43,948 OK209937 SPI-3, SPI-4 Proteus mirabilis (89%, MK422178.1 

N18S0357 100%) 

N18S0789 Derby SGI-1 44,852 OK209939 SPI-3, SPI-4 Proteus mirabilis (64%, MK422178.1 

100%) 

N16S319 Alachua SGI-1 63,305 OK209933 SPI-3, SPI-4 Proteus mirabilis (93%, KJ439039.1 

100%) 

F18S026 Senftenberg SGI-1 47,273 OK209932 SPI-3, SPI-4 Proteus mirabilis (86%, KJ439039.1 

100%) 

N17S834 Senftenberg SGI-1 117,891 OK209936 SPI-3, SPI-4 Citrobacter koseri (66%, CP026697.1 

89%) 

N18S0175 Saintpaul SGI-1 29,745 OK209938 SPI-3, SPI-4 Proteus mirabilis (97%, KJ439039.1 

100%) 

F18S010 I 4,[5],12:i:- SGI-4 81,780 MN730129.1 SPI-4, SPI-6 Citrobacter sp. (45%, 97%) CP056647.1 

F18S014 F18S032 F18S040 F18S043 F16S144 N17S056 N17S1466 F18S030 N18S0173 N16S319 Alachua SGI-4 ∼85,000 OK209934 SPI-4, SPI-6 Enterobacter hormaechei CP042551.1 

(97%, 100%) 
Li et al. Genomic Diversity of MDR Salmonella enterica 

TABLE 2 | Putative integrated plasmids in Salmonella isolates. 

ID Serotype Estimated size of Location of AMR and HMR on inserted Match with reference Plasmid types 

plasmid on insertion plasmid plasmid (accession No) 

chromosome 

F18S002 F18S010 I 4,[5],12:i:- 16 kb SPI-1, SPI-9 tet(B) merR merT merP merC pHCM1 (CP029645.1) IncQ 

F18S014 F18S032 sul2 aph(300

)-Ib aph(6)-Id 

F18S040 F18S043 blaTEM−1 

F18S045 N16S144 

N17S107 N17S380 

N17S1466 

N18S0173 

N17S146 I 4,[5],12:i:- 12 kb SPI-1, SPI-9 sul2 merC merP merT merR pHCM1 (CP029645.1) IncQ 

tet(B) 

F18S001 Muenster 31.6 kb SPI-5, SPI14 silE silS silR silC silF silB silA pF18S044-1 (ready for IncHI2 IncHI2A 

silP pcoA pcoB pcoC pcoD submission) 

pcoR pcoS pcoE 

F18S023 Johannesburg 31.6 kb SPI-6, SPI14 SilE silS silR silC silF silB silA pF18S044-1 (ready for IncHI2 IncHI2A 

silP pcoA pcoB pcoC pcoD submission) 

pcoR pcoS pcoE 

N17S0834 Senftenberg 31.6 kb SPI-3, SPI-8 SilE silS silR silC silF silB silA pF18S044-1 (ready for IncHI2 IncHI2A 

F18S026 silP pcoA pcoB pcoC pcoD submission) 

N18S0991 pcoR pcoS pcoE 

N18S0017 Agona 31.6 kb SPI-3, SPI-8 SilE silS silR silC silF silB silA pF18S044-1 (ready for IncHI2 IncHI2A 

silP pcoA pcoB pcoC pcoD submission) 

pcoR pcoS pcoE 

F18S033 Heidelberg 31.6 kb SPI-5, SPI14 SilE silS silR silC silF silB silA pF18S044-1 (ready for IncHI2 IncHI2A 

silP pcoA pcoB pcoC pcoD submission) 

pcoR pcoS pcoE 

N17S1304 Schwarzengrund 31.6 kb SPI-3, SPI-13 silE silS silR silC silF silB silA pF18S044-1 (ready for IncHI2 IncHI2A 

N18S1602 silP pcoA pcoB pcoC pcoD submission) 

pcoR pcoS pcoE 

F18S034 Derby 77.3 kb SPI-1, SPI-3 SilE silS silR silC silF silB silA pF18S029-1 (ready for IncHI2 IncHI2A 

silP pcoA pcoB pcoC pcoD submission) 

pcoR pcoS pcoE tet(A) merR 

merT merP bleO 

F18S013 Typhimurium 71 kb SPI-5, SPI14 blaCMY−2 pF18S007-1 (ready for IncI 

submission) 

N16S098 Heidelberg 71 kb SPI-6, SPI-16 blaCMY−2 pF18S007-1 (ready for IncI 

submission) 

F18S033 Heidelberg 54 kb SPI-4, SPI-6 qacEdelta1 cmlA5 ant(200)-Ia p24358-2 (CP051360.1) IncC 

tet(A) aph(6)-Id aph(300)-Ib 

sul2 

N18S0597 Typhimurium 85 kb SPI-1, SPI-9 terW terZ terD tet(C) aadA1 pSDC-F2_12BHI2 IncHI2 IncHI2A 

N18S1595 aac(3)-VIa qacEdelta1 sul1 (MH287085.1) 

N18S2170 merE merD merA merT merR 

silE silS silR silC silF silB silA 

silP 

N18S0981 Typhimurium 76.4 kb SPI-1, SPI-9 terW terZ terD tet(C) aadA1 pSDC-F2_12BHI2 IncHI2 IncHI2A 

aac(3)-VIa qacEdelta1 sul1 (MH287085.1) 

merE merD merA merT merR 

silE silS silR silC silF silB silA 

silP 

N18S0597 Typhimurium 107 kb SPI-6, SPI-16 merR tet(A) sul2 pN18S1634-2 IncC 

N17S0520 

N18S0666 

N18S0981 

N18S2170 

N17S0520 Typhimurium 83.5 kb SPI-6, SPI-16 merR tet(A) sul2 pN18S1634-2 IncC 

N18S0666 

N18S0981 

N18S2170 

(Continued) 

Frontiers in Microbiology | www.frontiersin.org 7 November 2021 | Volume 12 | Article 777817 
; 't Hi ~m .................... flK 'OIJ Typhimurium 

.. _ ~l~SOIII 4,(5),12:i:-

-----lncQ -------

Li et al. Genomic Diversity of MDR Salmonella enterica 

TABLE 2 | (Continued) 

ID Serotype Estimated size of Location of AMR and HMR on inserted Match with reference Plasmid types 

plasmid on insertion plasmid plasmid (accession No) 

chromosome 

N18S1595 Typhimurium 56 kb SPI-6, SPI-16 merR tet(A) sul2 pF18S004 Unknown - not closed 

N17S0166 I 4,[5],12:r:- 158 kb SPI-1, SPI-9 pcoS mcr-9.1 arsC aadA1 pN53053 (CP049311.1) IncHI2 IncHI2A 

aac(3)-VIa qacEdelta sul1 

N18S0736 I. 4,[5],12:r:- 38 kb SPI-1, SPI-9 sul1, qacEdelta1, aac(3)-VIa, pN53053 (CP049311.1) IncHI2 IncHI2A 

aadA1, arsR 

F18S004 Typhimurium 21 kb SPI-6, SPI-9 tetW tetZ tetD pF18S044-1 IncHI2 IncHI2A 

F18S013 Typhimurium 3.8 kb SPI-5, SPI-14 blaCMY−2 pF18S003-1/pF18S007- IncC/IncI/IncB/O/K/Z 

1/pN16S065-2 

N16S021 N16S070 Typhimurium 3.8 kb SPI-9, SPI-12 blaCMY−2 pF18S003-1/pF18S007- IncC/IncI/IncB/O/K/Z 

N16S089 N16S098 1/pN16S065-3 

N16S189 N16S214 

N18S0645 

18S1677 

N18S2188 

N18S0406 Bredeney 3.8 kb SPI-3, SPI-13 blaCMY−2 pF18S003-1/pF18S007- IncC/IncI/IncB/O/K/Z 

1/pN16S065-10 

F18S049 Hadar 15.0–19.3 kb SPI-2, SPI-12 aph(300)-Ib aph(6)-Id tet(A) pH1038-142(KJ484634.1) IncN IncFII 

N17S1270 

N18S1943 

N18S2154 

N18S2042 Infantis 91 kb SPI-2, SPI-12 tet(A) merR merT merP merC pN16S024 (CP052840.1) IncHI2 

blaCTX−M−65 

FIGURE 2 | IncQ plasmid integrated into Salmonella I 4,[5],12:i:- chromosome. The circle on the left represents a chromosome structure of a S. Typhimurium strain 

F18S013 with SPIs (in red) distributed on the chromosome. The green area was the sequence homologous to a Salmonella I 4,[5],12:i:- strain F18S010. The gray 

area shows differences between the two strains. The detailed comparison of the genomic structure of the two strains on the right is from the area with black box on 

the chromosome. 

N18S0173, and N18S2170) carried multiple chromosomal ARGs 

(Supplementary Table 1). These isolates encompassed seven 

dierent serotypes. 

Chromosomal HMRGs were found in 51 of 134 isolates, 

including genes encoding resistance to copper, silver, mercury, 

and arsenic. These comprised 22 turkey, 14 swine, 11 chicken, 

and 4 cattle isolates. Thirty-nine of the ffty-one isolates with 

more than three chromosomal HMRGs also had chromosomal 

ARGs. In many cases, HMRGs and ARGs were physically 

linked (Table 2). 

Together these fndings among 134 Salmonella genomes 

show that the maintenance and spread of chromosomal 

ARGs and HMRGs in Salmonella is accomplished through 

a complex interplay of genomic islands and integrated 

plasmids. Further work will be needed to understand 

whether acquisition of these genes is specifcally selected 

for by exposure to heavy metals and/or antimicrobials or 

connected to other survival and ftness challenges faced 

by Salmonella. 

Plasmid Types and Association With 

Resistance Genes, Sources, and 

Serotypes 

From the 134 Salmonella isolates we developed high-quality 

sequences for 285 plasmids, 245 of which we fully resolved into 

circular structures. On average there were two plasmids per 

isolate, although seven isolates had no plasmids and one had 

Frontiers in Microbiology | www.frontiersin.org 8 November 2021 | Volume 12 | Article 777817 
"' 

70 

60 

~ 50 

E 

"' -a_ 40 

-0 

t 30 .c 

E 

~ 20 

10 

0 

Ince 

Retail chicken 

Retail beef 

■ Cecal chicken 

■ Cecal cattle 

lncF lnrH 

Retail turkey 

Retail pork 

■ Cecal turkey 

■ Cecal swine 

Incl lncQ 

Replicon type 

lncX Col Multiple 

Li et al. Genomic Diversity of MDR Salmonella enterica 

FIGURE 3 | Plasmid types present across different food animal sources. Replicon information was based on analysis from PlasmidFinder. Only the most common 

plasmid types are represented in the fgure. 

TABLE 3 | Association of plasmid types with antimicrobial resistance genes (ARGs) and heavy metal resistant genes (HMRGs). 

Plasmid types Total number Avg. size in kb (range) Most common serotype (number) Avg. ARGs (range) Avg. HMRGs (range) 

IncA 2 133 (90–176) Heidelberg, I. 4,[5],12:i:- (1) 5.5 (5–6) 2.5 (0–5) 

IncB/O/K/Z 1 115 Kentucky 4 4 

IncC 36 142 (52–232) Typhimurium (11) 6.6 (0–13) 3.8 (0–8) 

IncF 23 112 (16–164) Kentucky (9) 2.1 (0–6) 0.8 (0–7) 

IncH 15 270 (145–354) Kentucky (3) 5.4 (1–14) 13.9 (3–22) 

IncI 39 99 (53–125) Kentucky, I. 4,[5],12:i:- (7) 1.6 (0–4) 0.7 (0–15) 

IncN 3 57 (43–71) Enteritidis, Heidelberg, I 4,[5],12:i:- 3 (1–5) 0 

IncP 1 18 I 4,[5],12:i:- 2 0 

IncQ 11 11 (8–12) Reading (5) 3.3 (1–4) 0 

IncR 1 70 Muenster 10 0 

IncX 22 42 (31–53) Kentucky (9) 0.4 (0–6) 0 

IncY 1 92 I. 4,[5],12:i:- 0 0 

Col 69 5 (2–15) Typhimurium (11) 0.3 (0–2) 0 

Phage-like 1 91 Typhimurium 0 0 

Combination 12 237 (75–389) Infantis (4) 6.3 (0–12) 6.3 (0–18) 

Unknown (no replicon) 28 18 (1–186) Typhimurium (5) 0.6 (0–6) 0.5 (0–14) 

Chromosome 134 4.8 Mb (4.5–5.1 Mb) N/A 1.7 (0–7) 7.3 (0–25) 

as many as seven plasmids, based on closed, circular sequences 

(Supplementary Table 1). We sought to identify the AMR and 

HMRGs on these plasmids and evaluated if any plasmids had 

animal source or serotype-specifcity. Although some plasmid 

types were most common in certain sources, most were widely 

distributed across all animal sources (Figure 3); some association 

between plasmid type and serotype was also noticed (Table 3). 

IncC

Sizes of the 36 IncC plasmids varied considerably, from 52 to 

232 kb, and were found among all animal sources (Table 3). These 

plasmids were found among 11 dierent serotypes, with the 

most prevalent being S. Typhimurium. Some common resistance 

patterns emerged, and 34 of the plasmids had sul2 and tetA, and 

22 had all of blaCMY−2, foR, and aph(6)-Id/aph(300)-Ib. Twelve of 

the isolates also had at least one qac gene and 29 had at least one 

mercury resistance gene. 

IncHI

There were 15 IncHI plasmids identifed among twelve dierent 

serotypes, present in all food animal sources, with swine being 

most common (7/15, 47%). All were large plasmids of 145– 

354 kb and possessed from one to fourteen ARGs. Seven also had 

the qacE biocide resistance gene, and all had at least one metal 

resistance operon, including those conferring resistance to silver, 

copper, mercury, and arsenic. 

IncI

There were 39 IncI plasmids in our collection of 134 genomes, 

from 19 dierent serotypes, with at least one ARG found in 33 of 

Frontiers in Microbiology | www.frontiersin.org 9 November 2021 | Volume 12 | Article 777817 
Li et al. Genomic Diversity of MDR Salmonella enterica 

the plasmids. They ranged in size from 62 to 125 kb and were 

found from all four food animal sources. The most common 

ARG was the clinically relevant blaCMY−2, which was found in 

15 isolates, including six S. Kentucky from retail chicken. Only 

two of the plasmids had HMRGs, and the qacE gene was seen 

in eight of the plasmids. The most common animal source for 

IncI plasmids was turkey, with 22 isolates, and 6 of these were in 

serotype I 4,[5],12:i:-. 

Other Plasmid Types

Several other plasmid types were identifed, including Inc R, 

A, B/O/K/Z, Q, N, P, F, and Y in descending ARG prevalence, 

respectively. The 27 IncF plasmids most commonly encoded 

either no ARGs (10/27) or only aminoglycoside and tetracycline 

resistance genes (8/27). Only 4/15 IncX plasmids encoded ARGs. 

Small Col-type plasmids contained few ARGs, with only 7/72 

encoding any ARGs, spread widely across dierent sources 

(Figure 3). There were 11 IncQ plasmids (aside from those 

integrated into the chromosome) found among diverse serotypes 

and sources, nine of which had the genes sul2, aph(6)-Id/aph(300)-

Ib, and tetA. There were also three IncN plasmids, two IncA, and 

one plasmid each of IncP, IncR, IncY, and IncB/O/K/Z. 

Twelve plasmids had multiple replicon types, indicating likely 

recombination between multiple plasmid types. These included 

IncHI, IncC, IncI, IncN, IncF, IncP, IncQ, and IncX replicons 

(Supplementary Table 1). All but one of these hybrid plasmids 

had ARGs for at least three dierent drug classes. 

A total of 44/285 plasmids did not have hits based on 

PlasmidFinder, indicating a failure of conventional typing 

techniques to identify them. Even though these plasmids were 

un-typeable, three contained ARGs (Supplementary Table 1). 

Overall, there were 33 Salmonella serotypes represented, with 

some interesting serotype-specifc fndings. Serotype Agona had 

blaCMY−2 as part of multidrug-resistant IncC plasmids in six of 

ten isolates. Similarly, MDR IncC plasmids were found in all four 

S. Montevideo and all fve S. Newport isolates. 

Serotype Kentucky also had IncX1 plasmids in ten of eleven 

isolates, with each of these being from chicken sources. There 

was one S. Kentucky isolate from turkey which did not have 

this plasmid and it did not possess any ARGs (Supplementary 

Table 1). Seven S. Kentucky isolates also had IncF plasmids 

with aph(30

)-Ib/aph(6)-Id, and tet(A); this combination of 

plasmid type and resistance genes was not observed in any 

other serotypes. 

Other serotypes also had specifc plasmid/resistance gene 

combinations. For instance, fve of six S. Reading isolates had 

IncQ plasmids with the ARGs aph(30)-Ib/aph(6)-Id, sul2, and 

tet(A), among only nine total isolates with this combination of 

plasmid type and resistance genes. All but one of these nine 

isolates were from turkey sources. Of importance to Salmonella 

virulence is that spv operon were found in nine isolates of four 

serotypes, including fve Typhimurium, one I 4,[5],12:i:-, two 

Dublin, and one Enteritidis. IncF (IncFII(S) subtype) replicons 

were present in all but one plasmid with spv operon, with one 

missing this replicon likely because it was not fully circularized. 

IncC and IncHI plasmid types were most strongly associated with 

ARGs (Table 3). 

Some genes that were plasmid-specifc include catA and mcr9 only on IncHI plasmids (Tyson et al., 2020) and qnrB19 only 

on Col plasmids. Other genes, such as blaTEM−1 and tet(B), were 

more widely distributed across dierent plasmid types (Figure 4). 

Overall, there was an association between the presence of ARGs 

and the presence of HMRGs on the same plasmid (Figure 5). 

DISCUSSION 

Here we present the results from long-read sequencing of 134 

Salmonella genomes, including over two hundred plasmids with 

circularized sequences. This sequencing eort produced several 

important fndings, including insight into the acquisition of 

virulence and AMR determinants. 

The size of SPIs ranged from 1.7 to 133.3 kb, encoding 1–21 

virulence genes. A phylogenetic tree based on the presence and 

absence of genes in each SPI from our previous study showed 

that, aside from serotype Choleraesuis, many other serotypes 

showed great diversity, particularly Newport, Derby, Agona, 

Anatum, and Typhimurium (Zhao et al., 2020). Some SPIs were 

more conserved across serotypes, such as SPIs 1-5, which had 

≥96% identity and 72% length alignment with the reference SPI 

in all isolates. Some SPIs had greater variability, such as SPI6, which in some cases had 97% identity but only 21% length 

alignment with the reference SPI, although its position relative to 

other SPIs was consistent in all isolates. Despite the great diversity 

of SPIs, the virulence genes are highly specifc to individual 

SPIs. The presence of SPIs varied in dierent serotypes but was 

largely consistent within individual serotypes (Figure 1). Several 

publications showed dierent SPI profles by using dierent 

methods to identify the presence of SPIs (Amavisit et al., 2003; 

Suez et al., 2013). In this study, a combined method of presence 

of virulence gene, genomic location and SPI reference structures 

was used to identify the SPIs. 

Rarely, some virulence genes were found in multiple location, 

for example, gtrA genes were shared by both SPI-16 and SPI17. Since the genomic location of each SPI is conservative, 

the presence of SPI-16 or SPI-17 were easily decided by this 

combined method. 

In this study, it was found that the relative position of 

SPIs to each other was generally stable. Even SPIs that are in 

phylogenetically distant serotypes, such as SPI-18 in Kiambu 

and Bredeney, had conserved insertion locations. This was 

true even though the number of SPIs was variable across 

dierent serotypes, indicating a key role for the SPIs in the 

evolution of serotypes. 

The presence of additional elements was also able to be easily 

identifed in reference to the SPI positions, as shown in Figure 1. 

The conserved SPI genomic locations can help to identify large 

insertions, such as SGIs or other accessory elements. In all cases 

the combinations of SPIs were consistent within serotypes, which 

can potentially be used to orient assemblies with short-read data, 

acting as a scaold to understand the arrangement of the genome. 

Recent research shows that SGI-1 is an integrative mobile 

element that plays an important role in introducing antibiotic 

resistance in various Gram-negative bacteria, including 

Frontiers in Microbiology | www.frontiersin.org 10 November 2021 | Volume 12 | Article 777817 
40 

35 lncA lncB/O/K/Z Ince lncF 

lncH a Incl ■ lncN lncR 

,::i "'30 lncX ■ lncY Col ·e 

"' n, 25 

1i 

'o 20 ... 

.Cl "' 15 

E 

::, 

z 10 

5 

0 

aac(3)-IV b/aCMY-2 b/aTEM-1 

-

catA floR mcr-9 

■ qnr819 

■ tet{BJ 

Resistance gene 

160 

140 

"' 120 ■ Has metal genes cu 

.. 

IV 

0 100 No metal genes 

.!!! 80 -0 

cu 60 ..a 

E 

:, 40 

z. 

20 

0 - 0-1 2-4 ~5 

Number of AMR genes 

Li et al. Genomic Diversity of MDR Salmonella enterica 

FIGURE 4 | Plasmid-resistance gene associations. The information above relates to plasmids with typing information and does not include those with zero or 

multiple replicon types. 

FIGURE 5 | Correlation between presence of antimicrobial resistance genes (ARGs) and heavy metal resistant genes (HMRGs). This depicts information related to 

plasmids and the presence of these genes. 

S. enterica, Proteus mirabilis, and Acinetobacter baumannii 

(Cummins et al., 2020). We identifed twelve isolates with SGI-1 

sequences, either by homology to the reference SGI-1 sequence 

or from insertions in the same region. All 12 SGIs were similar to 

SGIs in Proteus mirabilis, but their close relatives also included 

sequences from Citrobacter and Enterobacter. This fnding 

further helped us to understand how these genomic islands were 

horizontally acquired (Table 1). 

We had other novel fndings, including an SGI-1 sequence 

in serotype Alachua and large SGI-1s of dierent origin in 

serotypes Senftenberg, and Saintpaul. Those SGI-1 had almost 

no homology to previously reported SGI-1. The great diversity 

made it impossible to name the variants alphabetically as typical 

approach. In this study we also resolve the issue associated with 

the naming of SGI sequences by proposing a new approach based 

on their relative position in the genome. For instance, SGI-0 and 

SGI-2 (Levings et al., 2008; de Curraize et al., 2020) as previously 

name can all named as SGI-1 based on their consistent positions 

with other SGI-1 variants. An additional example of SGI diversity 

is shown by a novel SGI island containing HMRGs in S. Alachua. 

Even though it has limited homology with a previously reported 

SPI-4 (95% identity and 40% length), it was named as SGI-4 

variant because of its genomic location. By naming SGIs based on 

location, we hope for a streamlined process for SGI nomenclature 

in future work as diverse SGI sequences are identifed. It can 

help to identify the potential new variants. It would be interesting 

to further investigate the prevalence and distribution of SGIs in 

Salmonella isolated from other sources, including from human 

and sick animals. In this study, we also found ARGs and 

HMRGs on many chromosomal sequences with 100% homology 

to plasmids, indicating fragment of plasmid integration into the 

chromosome (Table 2). In fourteen isolates of S. I 4,[5],12:i:-, 

MDR IncQ plasmids were inserted into the same location in 

the chromosome (Figure 2). This monophasic serotype often 

Frontiers in Microbiology | www.frontiersin.org 11 November 2021 | Volume 12 | Article 777817 
Li et al. Genomic Diversity of MDR Salmonella enterica 

results from dierent insertions, deletions, or other disruptions 

of the fjB gene in serotype Typhimurium (Yang et al., 2015). We 

believe our results are the frst to identify insertion of IncQ at 

this site and may have resulted in S. Typhimurium conversion to 

serotype I 4,[5],12:i:-. 

As expected, most AMR was mediated by plasmids. A total 

of 147 plasmids had one or more ARGs compared to only 74 

chromosomes, some of which carried integrated plasmids as 

well as genomic islands. Although there were certain plasmid 

type/resistance gene associations found only in particular 

serotypes, most were not typically source or serotype-specifc 

and carried diverse plasmid types linked with dierent AMR 

genes and HMRGs. In addition, we found that 60% MDR 

(>3 AMRGs) strains also carried >3 HMRGs, including those 

conferring resistance to copper, gold, mercury, silver, arsenic, 

and tellurium (Supplementary Table 1 and Figure 5). This coexisting of AMRs and HMRs is of interest as the presence of 

any of these metals in food animal production have the potential 

to co-select for AMR. This is of particular signifcance as these 

congregated HMRGs were found on newly discovered SGI-4 and 

plasmids conferring resistance to three or more antimicrobial 

classes (Figure 5). 

There were some limitations in this study. Only 134 

Salmonella isolates were sequenced, they were exclusively from 

food animals and retail meats, and the isolates were not randomly 

chosen. As a result, fndings from this study may not be broadly 

applicable to all Salmonella serotypes or genomes from dierent 

animals, foods, or environments. In addition, we focused our 

sequencing on multidrug-resistant isolates, so some plasmids 

found to be frequently associated with AMR may have lower 

associations in a broader context of Salmonella serotype or 

genomic diversity. Also, our work highlights a drawback of 

using incompatibility typing to identify plasmid types, as some 

plasmids often have either multiple replicon sequences or none. 

Furthermore, isolates with the pESI plasmid in S. Infantis 

were only identifed with IncF replicons, despite the fact that 

this plasmid resulted from a combination of multiple plasmid 

types (Tyson et al., 2021). Given these challenges, alternative 

approaches such as the use of Plasmid Taxonomic Units could 

help address at least some of these issues (Redondo-Salvo et al., 

2020). Despite the limitations, this study represents perhaps 

the largest collection of closed Salmonella genomes reported to 

REFERENCES 

Abebe, E., Gugsa, G., and Ahmed, M. (2020). Review on major food-borne zoonotic 

bacterial pathogens. J. Trop. Med. 2020:4674235. 

Amavisit, P., Lightfoot, D., Browning, G. F., and Markham, P. F. (2003). 

Variation between pathogenic serovars within salmonella pathogenicity islands. 

J. Bacteriol. 185, 3624–3635. doi: 10.1128/JB.185.12.3624-3635.2003 

Antipov, D., Hartwick, N., Shen, M., Raiko, M., Lapidus, A., and Pevzner, P. A. 

(2016). Plasmidspades: assembling plasmids from whole genome sequencing 

data. Bioinformatics 32, 3380–3387. 

Arai, N., Sekizuka, T., Tamamura, Y., Kusumoto, M., Hinenoya, A., Yamasaki, S., 

et al. (2019). Salmonella genomic island 3 is an integrative and conjugative 

element and contributes to copper and arsenic tolerance of salmonella enterica. 

Antimicrob. Agents Chemother. 63, e00429–19. doi: 10.1128/AAC.00429-19 

date and advances our understanding of Salmonella genomics 

including its genomic plasticity and evolution. 

Future work to evaluate the applicability of long-read data 

to short-read datasets, including the use of reference-assisted 

assemblies, will increase the level of detail in genome-based AMR 

surveillance such as that done in NARMS and other national 

surveillance programs. Greater eorts to close Salmonella 

genomes also will help improve our understanding of genomic 

plasticity, evolution, and virulence. This work will help refne 

risk assessments by revealing the associations of resistance and 

virulence on mobile DNA elements, making it possible to be more 

precise in targeted interventions to limit the spread of the most 

problematic Salmonella serovars, including those less likely to 

respond to antimicrobial therapy. 

DATA AVAILABILITY STATEMENT 

The datasets presented in this study can be found in 

online repositories. The names of the repository/repositories 

and accession number(s) can be found in the article/ 

Supplementary Material. 

AUTHOR CONTRIBUTIONS 

CL, GHT, and SZ: conceptualization of ideas, validation, formal 

analysis, investigation, and writing—original draft preparation. 

CL, GHT, C-HH, and SZ: methodology. CL, C-HH, and ES: 

software. ES, GET, UD, and PM: resources. CL and T-TT: data 

curation. CL, GHT, C-HH, LH, ES, T-TT, GET, UD, PM, and SZ: 

writing—review and editing. CL, GHT, and C-HH: visualization. 

ES, PM, and SZ: supervision. ES and PM: project administration 

and funding acquisition. All authors have read and agreed to the 

published version of the manuscript. 

SUPPLEMENTARY MATERIAL 

The Supplementary Material for this article can be found 

online at: https://www.frontiersin.org/articles/10.3389/fmicb. 

2021.777817/full#supplementary-material 

Bearson, B. L., Trachsel, J. M., Shippy, D. C., Sivasankaran, S. K., Kerr, B. J., Loving, 

C. L., et al. (2020). The role of salmonella genomic island 4 in metal tolerance 

of salmonella enterica serovar i 4,[5],12:I:- pork outbreak isolate usda15wa-1. 

Genes 11:1291. 

Blondel, C. J., Jimenez, J. C., Contreras, I., and Santiviago, C. A. (2009). 

Comparative genomic analysis uncovers 3 novel loci encoding type six secretion 

systems dierentially distributed in salmonella serotypes. BMC Genomics 

10:354. doi: 10.1186/1471-2164-10-354 

Boyd, D. A., Peters, G. A., Ng, L., and Mulvey, M. R. (2000). Partial characterization 

of a genomic island associated with the multidrug resistance region of 

salmonella enterica typhymurium dt104. FEMS Microbiol. 189, 285–291. doi: 

10.1111/j.1574-6968.2000.tb09245.x 

Branchu, P., Charity, O. J., Bawn, M., Thilliez, G., Dallman, T. J., Petrovska, L., et al. 

(2019). Sgi-4 in monophasic salmonella typhimurium st34 is a novel ice that 

Frontiers in Microbiology | www.frontiersin.org 12 November 2021 | Volume 12 | Article 777817 
Li et al. Genomic Diversity of MDR Salmonella enterica 

enhances resistance to copper. Front. Microbiol. 10:1118. doi: 10.3389/fmicb. 

2019.01118 

Carattoli, A. (2013). Plasmids and the spread of resistance. Int. J. Med. Microbiol. 

303, 298–304. doi: 10.1016/j.ijmm.2013.02.001 

Carattoli, A., Zankari, E., Garcia-Fernandez, A., Voldby Larsen, M., Lund, O., Villa, 

L., et al. (2014). In silico detection and typing of plasmids using plasmidfnder 

and plasmid multilocus sequence typing. Antimicrob. Agents Chemother. 58, 

3895–3903. doi: 10.1128/aac.02412-14 

Cheng, R. A., Eade, C. R., and Wiedmann, M. (2019). Embracing diversity: 

dierences in virulence mechanisms, disease severity, and host adaptations 

contribute to the success of nontyphoidal salmonella as a foodborne pathogen. 

Front. Microbiol. 10:1368. doi: 10.3389/fmicb.2019.01368 

Cummins, M. L., Hamidian, M., and Djordjevic, S. P. (2020). Salmonella 

genomic island 1 is broadly disseminated within gammaproteobacteriaceae. 

Microorganisms 8:161. doi: 10.3390/microorganisms8020161 

de Curraize, C., Siebor, E., Neuwirth, C., and Hall, R. M. (2020). Sgi0, a relative 

of salmonella genomic islands sgi1 and sgi2, lacking a class 1 integron, 

found in proteus mirabilis. Plasmid 107:102453. doi: 10.1016/j.plasmid.2019. 

102453 

de Toro, M., Garcillaon-Barcia, M. P., and De La Cruz, F. (2014). Plasmid diversity 

and adaptation analyzed by massive sequencing of escherichia coli plasmids. 

Microbiol. Spectr. 2, 219–235. doi: 10.1128/microbiolspec.PLAS-0031-

2014 

Emond-Rheault, J. G., Hamel, J., Jeukens, J., Freschi, L., Kukavica-Ibrulj, I., 

Boyle, B., et al. (2020). The salmonella enterica plasmidome as a reservoir 

of antibiotic resistance. Microorganisms 8:1016. doi: 10.3390/microorganisms 

8071016 

Espinoza, R. A., Silva-Valenzuela, C. A., Amaya, F. A., Urrutia, I. M., Contreras, 

I., and Santiviago, C. A. (2017). Dierential roles for pathogenicity islands spi13 and spi-8 in the interaction of salmonella enteritidis and salmonella typhi 

with murine and human macrophages. Biol. Res. 50:5. doi: 10.1186/s40659-

017-0109-8 

Feldgarden, M., Brover, V., Haft, D. H., Prasad, A. B., Slotta, D. J., Tolstoy, I., 

et al. (2019). Validating the amrfnder tool and resistance gene database by 

using antimicrobial resistance genotype-phenotype correlations in a collection 

of isolates. Antimicrob. Agents Chemother. 63, e00483–19. 

Fookes, M., Schroeder, G. N., Langridge, G. C., Blondel, C. J., Mammina, C., 

Connor, T. R., et al. (2011). Salmonella bongori provides insights into the 

evolution of the salmonellae. PLoS Pathog. 7:e1002191. doi: 10.1371/journal. 

ppat.1002191 

Gardner, S. N., Slezak, T., and Hall, B. G. (2015). Ksnp3.0: snp detection and 

phylogenetic analysis of genomes without genome alignment or reference 

genome. Bioinformatics 31, 2877–2878. doi: 10.1093/bioinformatics/btv271 

Hall, R. M. (2010). Salmonella genomic islands and antibiotic resistance in 

salmonella enterica. Future Microbiol. 5, 1525–1538. doi: 10.2217/fmb.10.122 

Hayward, M. R., AbuOun, M., La Ragione, R. M., Tchorzewska, M. A., Cooley, 

W. A., Everest, D. J., et al. (2014). Spi-23 of s. Derby: role in adherence and 

invasion of porcine tissues. PLoS One 9:e107857. doi: 10.1371/journal.pone. 

0107857 

Hensel, M. (2004). Evolution of pathogenicity islands of salmonella enterica. Int. J. 

Med. Microbiol. 294, 95–102. 

Hsu, C. H., Li, C., Homann, M., McDermott, P., Abbott, J., Ayers, S., et al. 

(2019). Comparative genomic analysis of virulence, antimicrobial resistance, 

and plasmid profles of salmonella dublin isolated from sick cattle, retail beef, 

and humans in the united states. Microb. Drug Resist. 25, 1238–1249. doi: 

10.1089/mdr.2019.0045 

Ido, N., Lee, K., Iwabuchi, K., Izumiya, H., Uchida, I., Kusumoto, M., et al. (2014). 

Characteristics of salmonella enterica serovar 4,[5],12:I:- as a monophasic 

variant of serovar typhimurium. PLoS One 9:e104380. doi: 10.1371/journal. 

pone.0104380 

Jennings, E., Thurston, T. L. M., and Holden, D. W. (2017). Salmonella spi-2 

type iii secretion system eectors: molecular mechanisms and physiological 

consequences. Cell Host Microbe 22, 217–231. doi: 10.1016/j.chom.2017. 

Levings, R. S., Djordjevic, S. P., and Hall, R. M. (2008). Sgi2, a relative of salmonella 

genomic island sgi1 with an independent origin. Antimicrob. Agents Chemother. 

52, 2529–2537. doi: 10.1128/aac.00189-08 

Lou, L., Zhang, P., Piao, R., and Wang, Y. (2019). Salmonella pathogenicity island 1 

(spi-1) and its complex regulatory network. Front. Cell. Infect. Microbiol. 9:270. 

doi: 10.3389/fcimb.2019.00270 

Marcus, S. L., Brumell, J. H., Pfeifer, C. G., and Finlay, B. B. (2000). Salmonella 

pathogenicity islands: big virulence in small packages. Microbes Infect. 2, 145– 

156. doi: 10.1016/s1286-4579(00)00273-2 

McDermott, P. F., Tyson, G. H., Kabera, C., Chen, Y., Li, C., Folster, J. P., et al. 

(2016). Whole-genome sequencing for detecting antimicrobial resistance in 

nontyphoidal salmonella. Antimicrob. Agents Chemother. 60, 5515–5520. doi: 

10.1128/AAC.01030-16 

Partridge, S. R., Kwong, S. M., Firth, N., and Jensen, S. O. (2018). Mobile genetic 

elements associated with antimicrobial resistance. Clin. Microbiol. Rev. 31, 

e00088–17. 

Redondo-Salvo, S., Fernandez-Lopez, R., Ruiz, R., Vielva, L., de Toro, M., Rocha, 

E. P. C., et al. (2020). Pathways for horizontal gene transfer in bacteria revealed 

by a global map of their plasmids. Nat. Commun. 11:3602. doi: 10.1038/s41467-

020-17278-2 

Rychlik, I., Karasova, D., Sebkova, A., Volf, J., Sisak, F., Havlickova, H., et al. 

(2009). Virulence potential of fve major pathogenicity islands (spi-1 to spi-5) 

of salmonella enterica serovar enteritidis for chickens. BMC Microbiol. 9:268. 

doi: 10.1186/1471-2180-9-268 

Sabbagh, S. C., Forest, C. G., Lepage, C., Leclerc, J. M., and Daigle, F. (2010). 

So similar, yet so dierent: uncovering distinctive features in the genomes of 

salmonella enterica serovars typhimurium and typhi. FEMS Microbiol. Lett. 305, 

1–13. doi: 10.1111/j.1574-6968.2010.01904.x 

Scallan, E., Hoekstra, R. M., Angulo, F. J., Tauxe, R. V., Widdowson, M. A., 

Roy, S. L., et al. (2011). Foodborne illness acquired in the united states–major 

pathogens. Emerg. Infect. Dis. 17, 7–15. doi: 10.3201/eid1701.p11101 

Suez, J., Porwollik, S., Dagan, A., Marzel, A., Schorr, Y. I., Desai, P. T., et al. (2013). 

Virulence gene profling and pathogenicity characterization of non-typhoidal 

salmonella accounted for invasive disease in humans. PLoS One 8:e58449. doi: 

10.1371/journal.pone.0058449 

Tack, B., Vanaenrode, J., Verbakel, J. Y., Toelen, J., and Jacobs, J. (2020). Invasive 

non-typhoidal salmonella infections in sub-saharan africa: a systematic review 

on antimicrobial resistance and treatment. BMC Med. 18:212. doi: 10.1186/ 

s12916-020-01652-4 

Tate, H., Li, C., Nyirabahizi, E., Tyson, G. H., Zhao, S., Rice-Trujillo, C., et al. 

(2021). A narms survey of antimicrobial resistant foodborne bacteria isolated 

from retail veal in the united states. J. Food Prot. 84, 1749–1759. doi: 10.4315/ 

JFP-21-005 

Tyson, G. H., Li, C., Harrison, L. B., Martin, G., Hsu, C. H., Tate, H., et al. (2021). A 

multidrug-resistant salmonella infantis clone is spreading and recombining in 

the united states. Microb. Drug Resist. 27, 792–799. doi: 10.1089/mdr.2020.0389 

Tyson, G. H., Li, C., Hsu, C. H., Ayers, S., Borenstein, S., Mukherjee, S., et al. (2020). 

The mcr-9 gene of salmonella and escherichia coli is not associated with colistin 

resistance in the united states. Antimicrob. Agents Chemother. 64, e00573–20. 

doi: 10.1128/AAC.00573-20 

Vijayakumar, R., and Sandle, T. (2019). A review on biocide reduced 

susceptibility due to plasmid-borne antiseptic-resistant genes-special notes on 

pharmaceutical environmental isolates. J. Appl. Microbiol. 126, 1011–1022. doi: 

10.1111/jam.14118 

Worley, J., Meng, J., Allard, M. W., Brown, E. W., and Timme, R. E. (2018). 

Salmonella enterica phylogeny based on whole-genome sequencing reveals two 

new clades and novel patterns of horizontally acquired genetic elements. mBio 

9, e02303–18. doi: 10.1128/mBio.02303-18 

Yang, X., Wu, Q., Zhang, J., Huang, J., Guo, W., and Cai, S. (2015). Prevalence and 

characterization of monophasic salmonella serovar 1,4,[5],12:I:- of food origin 

in china. PLoS One 10:e0137967. doi: 10.1371/journal.pone.0137967 

Zhao, S., Li, C., Hsu, C. H., Tyson, G. H., Strain, E., Tate, H., et al. (2020). 

Comparative genomic analysis of 450 strains of salmonella enterica isolated 

from diseased animals. Genes 11:1025. doi: 10.3390/genes11091025 

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