Microbial Pathogenesis 173 (2022) 105808

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Microbial Pathogenesis
journal homepage: www.elsevier.com/locate/micpath

Genomic virulence genes profile analysis of Salmonella enterica isolates from

animal and human in China from 2004 to 2019
Shigan Yan , Xu Liu , Chengyu Li , Zhaoxu Jiang , Donghui Li , Liping Zhu *
School of Bioengineering, Qilu University of Technology (Shandong Academy of Sciences), Jinan, 250353, China

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

Keywords: Salmonella is a momentously zoonotic and food-borne pathogen that seriously threats human and animal health
Salmonella around the world. Salmonella pathogenicity is closely related to its virulence genes profile. However, conven­
Virulence genes tional virulence gene analysis methods cannot truly reveal whole virulence genes carried by Salmonella. In this
Pathogenicity
study, whole genome sequencing in combination with Virulence Factor Database were applied to investigate
Whole genome sequencing
whole virulence gene profiles of 243 Salmonella isolates from animals and humans in China from 2004 to 2019.
The results showed that a total of 670 virulence genes were identified in Salmonella, among them, 319 virulence
genes were found in all the Salmonella tested isolates, and 9 virulence genes were unique to Salmonella. The 670
virulence genes were classified into 14 categories according to their functions, and the genes related to adher­
ence, effector delivery system, immune modulation, motility and nutritional/metabolic factors accounted for
84.63%. Relationships between virulence genes and serovars, sequence types indicated that strains belonged to
the same serovar or sequence type had similar virulence genes profiles, however, isolates from different sources,
years and locations of isolation had variable virulence gene profiles. In addition, copy number of virulence genes
and homologous virulence genes shared with other pathogens were also analyzed in this study. In summary, we
investigated pan-genomic virulence gene profiles and molecular epidemiology of Salmonella isolates from
humans and animals in China from 2004 to 2019. These findings are beneficial for pathogenic monitoring,
investigation of virulence evolution as well as prevention and control of Salmonella.

1. Introduction
Salmonella is a momentously food-borne pathogens that can cause
typhoid, gastroenteritis, septicaemia, miscarriage and even death in
humans and animals [1]. Salmonella from animal-derived foods,
including primarily poultry, eggs, milk, pork and beef, has brought
about various degrees of disease load in humans [2]. As the second
leading food-borne disease, salmonellosis has become a global public
health hazard. Non-typhoid Salmonella is estimated to cause 93.8 million
cases of acute gastroenteritis and 155,000 deaths each year worldwide
[3]. Salmonella causes approximately 1.2 million illnesses and 450
deaths every year in the United States [4]. Salmonellosis represented the
second zoonotic disease in the European Union in 2020 [5]. Similarly,
Salmonella-caused illness accounts for 70%–90% of bacterial food
poisoning incidents in China [6,7].
Salmonella enterica subspecies enterica is responsible for the vast
majority of Salmonella infections in humans and warm-blooded animals
[8]. So far, more than 2600 distinct serovars have been identified based
on the somatic, flagellar and capsular antigen of Salmonella enterica [9].
Some serovars are host-specific and cause disease in restricted hosts, for
example, Typhi and Paratyphi only infect human, Gallinarum just in­
fects chickens; some serovars can infect a few kinds of hosts, for
example, Dublin can cause illness in cattle and humans; while others (e.
g., Typhimurium and Enteritidis) can contaminate a wide range of hosts.
Enteritidis and Typhimurium are predominant serovars of Salmonella
from humans all over the world [7]. Although wide application of
serological classification, the relationships between serovars and viru­
lence of Salmonella are still unclear.
The pathogenicity of Salmonella is closely associated with its viru­
lence genes [10]. Virulence genes are located at different sites of genome
and mobile genetic elements, including Salmonella pathogenicity islands
(SPIs), virulence plasmids, and bacteriophages. SPIs are compact and
unique regions of chromosomes that carry virulence genes. More than
twenty SPIs have been identified till now, which play different roles and
show variable prevalence in different serovars [11]. To date, a lot of
virulence genes in Salmonella have been determined, including

* Corresponding author.
E-mail address: zhuliping2012@aliyun.com (L. Zhu).

https://doi.org/10.1016/j.micpath.2022.105808
Received 4 June 2022; Received in revised form 7 September 2022; Accepted 26 September 2022
Available online 29 September 2022
0882-4010/© 2022 Elsevier Ltd. All rights reserved.
S. Yan et al.
adhesion-related genes (e.g. fim, lpf, bcf, stb, stc, std, stf, sth, sti, saf, stj
operons, shdA, misL, ratB, and sinH genes), the genes associated with
type III secretion system (e.g. inv, sip, spa, hil, prg, org, ssa, sse, ssr, ssc
operons), plasmid-coding genes (e.g. spv, pef, rck, mig-5) [9,12–16].
These genes coding proteins enable bacteria adapt host environment,
resist and overcome the host’s defensive immunity [17]. Virulence genes
profile analysis is beneficial for estimating bacterial potential
pathogenicity.
Conventional method for investigation of virulence genes is to
employ PCR amplification of several target virulence genes. Only a small
number of virulence genes can be detected simultaneously using this
low-throughput screening method. In addition, some strains that owned
the identical known virulence genes shew different pathogenicity,
indicating the presence or absence of some specific genes cannot fully
elucidate the pathogenesis of Salmonella. However, whole genome
sequencing (WGS) technology has revolutionized the pathogens sur­
veillance, bacterial identification, genotyping, antibiotic resistance and
virulence evaluation, as well as phylogenetic analysis [18]. With the
rapid advancement of high-throughput sequencing technology, public
phylogenetic resource databases emerge to comparative analysis of
numerous sequences. Among them, the Virulence Factor Database
(VFDB) is a widely-adopted web platform for virulence factor analysis of
pathogens [19]. The combination of WGS and VFDB aligning can
comprehensively investigate bacterial virulence genes, and thus deepens
the understanding of its pathogenicity.
In this study, we analyzed the virulence genes profiles of 243 strains
of Salmonella enterica from different sources in China from 2004 to 2019
based on WGS and VFDB aligning. These results provide a scientific
foundation for understanding the pathogenicity, molecular epidemics,
prevention and control of Salmonella.
2. Materials and methods
2.1. Salmonella strains and serovars
A total of 243 isolates were collected from multiple sources,
including poultry (n = 197), livestock (n = 15), and human patients (n
= 31). These strains were isolated from nine provinces in China from
Fig. 1. Background information of Salmonella isolates.
2
Microbial Pathogenesis 173 (2022) 105808
2004 to 2019, including Beijing (n = 55), Fujian (n = 11), Guangdong (n
= 13), Guangxi (n = 19), Henan (n = 16), Shaanxi (n = 63), Shandong
(n = 9), Shanghai (n = 29) and Sichuan (n = 28). All Salmonella enterica
isolates were identified using microbiological, biochemical and 16S
rDNA sequencing, and slide agglutination tests [20]. These tested strains
belonged to 36 serovars, including Enteritidis (n = 30), Typhimurium (n
= 29), Mbandaka (n = 22), Indiana (n = 21), Derby (n = 21), Thompson
(n = 17), etc. These 36 serovars were categorized into 10 serogroups,
namely B, C1, C2, C3, D1, E1, E4, G, I, K, and M. The serovars and
background information of these isolates were shown in Fig. 1 and
Table S1.
2.2. Whole genome sequencing and assembly
Genomic DNA of Salmonella strain was extracted using the Bacterial
Genome Extraction Kit (Tiangen, DP302-02), and the DNA quantity was
measured with MD2000H ultra-micro nucleic acid analyzer. The Sal­
monella genomes were sequenced on an Illumina Novaseq PE150 plat­
form using a 2 × 250 bp paired end (PE) reads for 500 cycles. The
sequence libraries were constructed using Illumina’s NEBNext Ultra
DNA Library Prep Kit (NEB, USA). The genomic sequence contigs were
assembled using SOAP de novo, SPAdes and AByss software, and then
integrated with CISA software. Finally, the assembly genomic sequences
with a minimum contig size threshold of 500 bp in length were obtained
using Gap Closer. Detailed operation steps referred to our previous
article [20].
2.3. In silico analysis of virulence genes in Salmonella based on whole
genomic sequences
The genomic sequences were aligned in the VFDB database (http://
www.mgc.ac.cn/VFs/) to obtain genomic virulence function annota­
tions [19]. In brief, amino acid sequences of the protein encoded by the
gene were aligned in the VFDB database using DIAMOND software with
default settings (identity≥40%; E-value≥1e-5) [21,22], and then the
annotation results were obtained, including the matched target gene and
the corresponding functional information.
S. Yan et al.
2.4. Statistic analysis of virulence genes
The detection rates of the virulence genes in each strain were sta­
tistically analyzed based on the annotation results.
Unique virulence genes to Salmonella were further screened from
those virulence genes with a detection rate of 100% in Salmonella using
BLAST search in the GenBank database (https://www.ncbi.nlm.nih.
gov/gene/). On the contrary, the homologous virulence genes shared
with other pathogens were also studied. In addition, copy number of the
genes with a detection rate of 100% in Salmonella was counted.
Moreover, the relationships between virulence genes and serovars,
sequence types (STs), as well as isolation sources, years or locations were
also investigated.
All data were analyzed and plotted using GraphPad Prism 9.0, and
the heat map was drawn using HemI software.
3. Results
3.1. Genomic information of 243 Salmonella enterica strains
The draft genome sequences of 243 Salmonella isolates tested in the
study were submitted to Sequence Read Archive (SRA) of NCBI, and the
SRA numbers obtained were summarized in Supplementary Table S1.
A total of 9490 contigs (>500bp) varying from 9 to 92 with an
average of 39 per genome were generated by sequencing. The average
draft genome size of the tested strain was 4.88 Mb, varying from 4.35 Mb
to 5.48 Mb. The average genomic G + C content was 52.04%, ranging
from 51.66% to 52.35%. Functional annotation predicated that all draft
genomes of Salmonella enterica isolates comprised of 4694 genes on
average (Table S2).
3.2. Detection rates analysis of virulence genes in Salmonella enterica
Through WGS and VFDB aligning analysis, a total of 670 virulence
genes in the 243 tested Salmonella enterica isolates were obtained, and
numbers of virulence genes existed in each strain ranged from 417 to
501. Among the 670 virulence genes, 319 virulence genes were present
in each one of the tested Salmonella isolates, while the other 351 viru­
lence genes only existed in part isolates with variable detection rates
(Fig. 2 and Table S3). In addition, 418 virulence genes appeared in more
than 80% of the isolates, 162 virulence genes were with the detection
rates less than 20%, and the remaining 90 virulence genes were found in
from 20% to 80% of the strains (Fig. 3).
Fig. 2. Heat map of 351 virulence genes present in 243 Salmonella isolates.
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Microbial Pathogenesis 173 (2022) 105808
3.3. Unique virulence genes in Salmonella
Among the 319 virulence genes with the detection rate of 100%, 9
virulence genes were firstly reported that they uniquely existed in Sal­
monella rather than other genera of bacteria (Table 1) through BLAST
search in the GenBank database. Most of these 9 genes locate on SPI-2
and their coding products that are mainly structural proteins and
secreted effector of the type III secretion system.
3.4. Functional category of virulence genes in Salmonella enterica isolates
The results of the functional classification of 670 virulence genes
using the VFDB database were shown in Table 2. All the 670 virulence
genes were divided into 14 categories, and among them, five categories,
namely adherence, effector delivery system, immune modulation,
motility and nutritional/metabolic factors, account for 84.63%. The
adhesion category mainly involved fimbriae- and adhesin-coding genes
that are closely related to the colonization of pathogens in the early
stage of infection. In the effector delivery system category, the genes
encoding type III secretory system and secretory effector proteins
covered the largest proportion, which either participate the formation of
the secretion system or being transported into the host cells to assist
Salmonella invasion and survival in host environment. Immune modu­
lation category mainly involves virulence genes encoding surface anti­
gens (e.g. capsules and lipopolysaccharides) to resist host immune
defenses and promote systemic spread. Flagella was the major virulence
factor in the motility category, it plays a role in promoting motility and
chemotaxis. The virulence genes encoding iron and magnesium trans­
porters, as well as biosynthesis-related enzymes, covered a major pro­
portion in nutrition and metabolism category, and their coding products
ensured the normal survival of Salmonella in the host. In addition, there
were also genes encoding two-component regulatory systems, extracel­
lular toxins, biofilm formation and stress proteins.
3.5. Copy number of 319 common virulence genes in Salmonella isolates
Copy number of 319 virulence genes with the detection rates of
100% in all the tested Salmonella isolates were furtherly investigated,
and the results were shown in Table 3. The copy number of different
genes were different, even the same genes in different isolates also
covered dissimilar copy number. 60 virulence genes owned single copy,
and 2 virulence genes (namely argK and bfmR) were with 2 copies. Copy
number of other genes varied in the tested strains: 212 genes with 1 or 2
copies, 22 genes with 1–3 copies, 2 genes (i.e. ctpV and YE105_C1500)
with 1–4 copies, 11 genes (i.e. algW, armR, CBU_1566, cheD, galU, hemN,
htrB, kpsF, mgtB, narH, sodB) with 2–3 copies, 3 genes (i.e. lafK, mtrD and
VVA1298) with 2–4 copies, 2 genes (oppF, pilR) with 2–5 copies, tar/
cheM with 3–5, pilW with 3–6, mprA with 3–7, ompD with 6–10, and sugC
with 7–10.
3.6. Relationships analysis between virulence genes and serovars, STs
We counted the average numbers of virulence genes in Salmonella
strains belonged to different serovars. Havana contained the most
number of 501, followed by Kentucky with 489 and Typhimurium with
484, while Stanley and Uganda had the least number of 433 and 428,
respectively, and we found most strains belonged to the same serovars
had similar number of virulence genes (Fig. 4). The results also showed
that the same Salmonella serovar strains had identical or similar viru­
lence genes, and some virulence genes were only presented in the cor­
responding serovars. For example, aec17, aec18, aec22, aec23, aec24,
aec25, aec27/clpV, aec29, aec30, aec31 and EC042_0221 were only in
Havana; btpA, ipaH7.8, prt, sefBCDR, YPK_3186 only in Enteritidis;
C8J_1080, M3Q_285, M3Q_287, M3Q_290, M3Q_291 only in Pomona;
cshE, mrxJ only in Schwarzengrund; manC only in Hvittingfoss; wbyK
only in Hvittingfoss; YpsIP31758_3038 only in Hvittingfoss; cylG only in
S. Yan et al. Microbial Pathogenesis 173 (2022) 105808

Fig. 3. Correlations between detection rates and the number of virulence genes.

serogroup B, C2, C3, D1, E1 and E4, wbcC only in serogroup B and D1,
Table 1
Ypk_3179 only in serogroup I, K and M.
The nine unique virulence genes in Salmonella.
Furthermore, the relationships between several crucial virulence
Virulence genes Gene location Gene description genes and serovars of Salmonella, including adhesion-related genes, the
sopD Chromosome type III secretion system effector SopD genes coding type III secretion system, plasmid encoding genes, were
sprB SPI-1 transcriptional regulator analyzed. The results showed that adhesion-related genes, (e.g. csg, bcf,
ssaB SPI-2 pathogenicity island secreted effector protein
fim, stb, sth operons and stdABC, steAC, ratB, shdA, sinH), were detected
ssaI SPI-2 type III secretion system apparatus protein
ssaO SPI-2 type III secretion system stalk protein SsaO in all serovars, and sefABCD was only present in Enteritidis, while the
ssaP SPI-2 type III secretion protein others were present in different serovars. Most of the T3SS-coding genes
sseE SPI-2 type III secretion system effector SseE existed in all 36 serovars, which indicated that these genes were highly
sseF SPI-2 type III secretion system effector SseF conserved in Salmonella. Among 11 virulence genes (i.e. spvR, spvA,
sthD Chromosome fimbrial protein SthD
spvB, spvC, spvD, pefA, pefB, pefC, pefD, mig-5, rck) encoded by plasmid in
this study, only rck was present in all serovars, and the other genes were
Weltevreden; EC958_0641 only in London; EC958_5138 only in Cor­ only detected in some strains of Enteritidis, Typhimurium Saintpaul and
vallis; faeJ only in Kentucky; ipaH1.4 only in Cerro; SSU98_1513 only in Havana. Some functional genes, such as Mg2+ transport protein coding
Tennessee; tcpC only in Anatum. genes mgtBC, macrophage inducing gene mig-14, iron uptake regulating
Moreover, some genes were only present in specific serogroups. For gene fur, transcription activator coding genes phoPQ, inner membrane
example, aec15 was only in serogroup G and K2, ddhABCD only in protein coding gene siiE and stress protein coding gene sodCI were
serogroup B, C2, C3 and D1, F7308_0881 and wbaP/rfbP only in present in all serovars, while the typhoid toxin encoding gene cdtB just

4
S. Yan et al. Microbial Pathogenesis 173 (2022) 105808

Table 2 Table 2 (continued )

The functional category of 670 virulence genes in Salmonella.
Functional category and
number of genes
Effector delivery system (192)
Adherence (163)
Immune modulation (98)
Motility (64)
Functional category and Virulence gene
Virulence gene number of genes
Nutritional/Metabolic factor basG, bioB, ccmB, ccmC, ccmE, ccmF, ctpV, entA,
ABB77406, ABB77417, aec15, aec17, aec18, aec19, (64) entB, entC, entD, entE, entF, entS, F7308_0432, fagC,
aec22, aec23, aec24, aec25, aec27/clpV, aec29, fbpC, feoA, feoB, fepA, fepB, fepC, fepD, fepE, fepG,
aec30, aec31, aec32, aec7, ASA_2456, avrA, btpA, fes, FN3523_0020, FN3523_0021, FNFX1_1207,
CBU_1566, CbuG_0575, cesT, clpB, clpV, cts2H, ctsD, Fphi_0805, hemA, hemB, hemC, hemD, hemE, hemG,
EC042_0221, EC55989_3316, EC958_0639, hemH, hemL, hemN, hitA, hitC, hpt, ireA, irgA, iroB,
EC958_0641, ehaA, espN2-2, glrA, gogB, grlR, hilA, iroC, iroD, iroE, iroN, kasB, leuD, mbtI, mgtB, mgtC,
hilC, hilD, hopAN1, hrpH, iacP, iagB, invA, invB, narG, narH, panC, panD, phuU, pvcC, sitA, sitB, sitC,
invC, invE, invF, invG, invH, invI, invJ, ipaH, sitD
ipaH1.4, ipaH2.5, paH7.8, lidL, lirB, lpg0021, Regulation (13) bfmR, cdpA, csrA, fur, gacA, mprA, perC/bfpW, phoP,
lpg2936, lpnE, mlr6325, mlr6326, nleK, phoQ, regX3, relA, rpoS, sigA/rpoV
O3M_04335, orgA, orgB, orgC, pipB, pipB2, prgH, Biofilm (12) adeG, adeF, algU, algW, cah, EC958_5138, farA,
prgI, prgJ, prgK, ricA, Sp0731, rvhB1, sciA, sciB, sciC, luxS, mucD, mucP, rhlR, upaH
sciD, sciE, sciF, sciH, sciI, sciJ, sciK, sciL, sciM, sciN, Stress survival (12) ibpA, ahpC, clpC, clpE, clpP, katA, katG, mntB, mntC,
sciO, sciP, sciQ, sciR, sciS/icmF-like, sciT, sciU, ciV, recN, sodB, sodCI
sciW, sfa3, SG1029, SG1030, SG1031, SG1032, Exotoxin (9) argK, B565_0799, cdtB, cylG, cysC, hlyA, SPA1306,
SG1033, SG1035, SG1036, SG1037, SG1038, spvB, STY1498
SG1039, SG1040, SG1041, SG1042, SG1043, Antimicrobial activity/ farB, mig-14, mig-5, mtrC, mtrD, mtrE
SG1044, SG1048, SG1049, sicA, sicP, sifA, sifB, sipA, Competitive advantage (6)
sipB, sipC/sspC, sipD, slrP, sopA, sopB/sigD, sopD, Invasion (5) ail, ibeB, ibeC, inv, traJ
sopD2, sopE, sopE2, spaO, spaP, spaQ, spaR, spaS, Exoenzyme (2) eno, pla
spiC/ssaB, sprB, sptP, spvC, spvD, ssaC, ssaD, ssaE, Post-translational mip, lptA
ssaG, ssaH, ssaI, ssaJ, ssaK, ssaL, ssaM, ssaN, ssaO, modification (2)
ssaP, ssaQ, ssaR, ssaS, ssaT, ssaU, ssaV, sscA, sscB, Other uncategorized genes armR, CAA68592, cib, clpL, ECS88_3547, fct, foxR,
sseA, sseB, sseC, sseD, sseE, sseF, sseG, sseI/srfH, sseJ, (28) glnA1, hasF, icl, ldaB, lpg1062, msbB2, pcaA, pdtorfF,
sseK1, sseK2, sseL, sspH1, sspH2, ssrA, ssrB, pdtorfM, PMI0229, PMI0230, pulO, pvuE, qbsC,
STM0278, tapD, trwD, trwE, trwK, trwM, tsh, vgrS, spvA, spvR, SSU98_1513, STM0570, vapA5, virK,
virB, virB1, virB11, VPA1380, YpsIP31758_B0107, VVA1298
YpsIP31758_B0108
afaB, afaC, afrS, air/eaeX, bcfA, bcfB, bcfC, bcfD,
bcfE, bcfF, bcfG, bcfH, CT396, cofT, comE/pilQ, csfB,
csfC, csfD, csfE, csfF, cshB, cshE, csgA, csgB, csgD, Table 3
csgE, csgF, csgG, csvA, ehaB, etpB, faeC, faeE, faeF, Copy number of 319 common virulence genes in all Salmonella isolates.
faeH, faeJ, fimA, fimB, fimC, fimD, fimF, fimH, fimI,
Copy Virulence genes
fimW, fimY, fimZ, Fphi_1039, hofB, hofC, htpB, IlpA,
number
lap, lpfA, lpfB, lpfC, lpfD, lpfE, misL, ML1683, mrxJ,
O3M_20930, ompA, ompD, pagN, pefA, pefB, pefC, 1 afrS, ahpC, ail, algU, bcfA, bcfB, bcfC, bcfD, bcfE, bcfF, bcfG, bcfH, bplF,
pefD, pegA, pegB, pegC, pegD, pilL, pilM, pilN, pilO, CbuG_0575, clpP, ECS88_3547, ehaB, F7308_0432, fleR/flrC, fliD, fliE,
pilP, pilQ, pilR, pilS, pilT, pilU, pilV, pilW, plr/gapA, fliF, fliG, fliH, fliI, fliJ, fliK, fliL, fliM, fliN, fliO, fliP, fliQ, fliR, fliS, fliT,
ppdD, psaE, ratB, rpoN, safA, safB, safC, safD, flk, foxR, glnA1, gmhA, hemB, ibeC, ibpA, katG, kdtA, kdtB, mrsA/glmM,
SeAg_B4896, SeAg_B4897, sefB, sefC, sefD, sefR, mtrE, ndk, opsX/rfaC, recN, regX3, rfaD, rfaF, rpoN, sinH, sseL, waaG,
shdA, siiE, sinH, staA, staB, staC, staD, staE, staF, waaP, YPA_2067
staG, stbA, stbB, stbC, stbD, stbE, stcA, stcB, stcC, 1–2 ABB77406, acpXL, adeF, adeG, bioB, cap8J, CFF8240_1412, cheA, cheB,
stcD, stdA, stdB, stdC, stdD, steA, steB, steC, steD, cheR, cheW, cheY, cheZ, clpC, cpsA, csgA, csgB, csgD, csgE, csgF, csgG,
steE, steF, stfA, stfC, stfD, stfE, stfF, stfG, sthA, sthB, csrA, cysC1, eno, entA, entB, entC, entD, entE, entF, entS, farA, farB,
sthC, sthD, sthE, stiA, stiB, stiC, stiH, stjA, stjB, stjC, feoA, feoB, fepA, fepB, fepC, fepD, fepE, fepG, fes, fimA, fimC, fimD, fimF,
stkA, stkB, stkC, stkD, stkE, stkF, stkG, STM4574, fimH, fimI, fimW, fimY, fimZ, fleQ/flrC, fleR, flgA, flgB, flgC, flgD, flgE,
STM4575, tcfA, tcfB, tcfC, tcfD, ugpB, upaC, upaG/ flgF, flgG, flgH, flgI, flgJ, flgK, flgL, flgM, flgN, flhA, flhB, flhC, flhD, flhE,
ehaG, vfr fliA, fliB, fliZ, FN3523_0020, FN3523_0021, FNFX1_1207, Fphi_0805,
AB57_0110, ABK1_0097, ABZJ_00085, fur, gacA, hasF, hemA, hemH, hemL, hisH2, hlyA, hofC, hopAN1, hpt,
ABZJ_00086, acpXL, adhD, BCAL3235, bplF, htpB, invA, invB, invC, invE, invF, invG, invH, invI, ipaH, iroB, iroC, iroD,
C8J_1080, cap8J, CFF8240_1412, cpsA, cpsF, ddhA, iroE, iroN, kasB, kdsA, leuD, lpg0021, lpg2936, lptA, lpxC, lpxH, lpxK,
ddhB, ddhC, ddhD, eps4, F7308_0881, fabZ, luxS, mbtH, mbtI, mgtC, mig-14, mip, motA, motB, msbA, msbB2, nueA,
FN3523_0439, Fphi_1467, galE, galU, gmd, gmhA, ompA, orfM, agP, panC, panD, pbpG, phoP, phoQ, phuU, pipB, plr/gapA,
gmhA/lpcA, gtrA, gtrB, hisH2, htrB, kdsA, kdtA, kdtB, ppdD, pvcC, rck, relA, rfaE, rhlR, rpoS, RSp0731, sicA, sifA, sigA/rpoV,
kpsF, lpxA, pxB, lpxC, lpxD, lpxH, lpxK, M3Q_285, sipA, sipB, sipC/sspC, sipD, sitA, sitB, sitC, sitD, sopA, sopB/sigD, sopD,
M3Q_286, M3Q_287, M3Q_290, M3Q_291, spaO, spaP, spaQ, spaR, spaS, spiC/ssaB, sprB, ssaC, ssaD, ssaE, ssaG,
M3Q_301, manB, manC, mbtH, mrsA/glmM, msbA, ssaH, ssaI, ssaJ, ssaK, ssaL, ssaN, ssaO, ssaP, ssaQ, ssaR, ssaS, ssaT, ssaU,
ndk, oppF, opsX/rfaC, orfH, orfM, PA3142, PA3144, ssaV, sscA, sscB, sseA, sseB, sseC, sseD, sseE, sseF, sseG, sseJ, ssrA, ssrB,
pagP, pbpG, pgi, prt, rck, rfaD, rfaE, rfaF, rmlA, rmlB, stdA, sthA, sthB, sthC, sthD, sthE, STM0570, ugpB, virK, wbfV/wcvB,
rmlC, sugC, tcpC, uppS, waaG, waaP, wbaP/rfbP, wcaG, wza, wzb, wzc, Z1307, pdtorfM
wbcC, wbfB, wbfV/wcvB, wbjD/wecB, wbyK, wcaG, 1–3 clpE, CT396, cts2H, fabZ, flmH, hitC, IlpA, lpxA, lpxB, lpxD, mtrC, mucP,
wcbN, wecA, wza, wzb, wzc, wzt, YE105_C0173, narG, pilT, ratB, rmlB, slrP, sodCI, steA, steC, uppS, wcbN
YE105_C1500, YE105_C1504, YPA_2067, 1–4 ctpV, YE105_C1500
YPA_2596, YPK_3178, YPK_3179, YPK_3186, 2 argK, bfmR
YpsIP31758_3038, Z1307 2–3 algW, armR, CBU_1566, cheD, galU, hemN, htrB, kpsF, mgtB, narH, sodB
cheA, cheB, cheD, cheR, cheW, cheY, cheZ, flaA, 2–4 lafK, mtrD, VVA1298
flbD, fleQ, fleQ/flrC, fleR, fleR/flrC, flgA, flgB, flgC, 2–5 oppF, pilR
flgD, flgE, flgF, flgG, flgH, flgI, flgJ, flgK, flgL, flgM, 3–5 tar/cheM
flgN, flhA, flhB, flhC, flhD, flhE, fliA, fliB, fliC, fliD, 3–6 pilW
fliE, fliF, fliG, fliH, fliI, fliJ, fliK, fliL, fliM, fliN, fliO, 3–7 mprA
fliP, fliQ, fliR, fliS, fliT, fliY, fliZ, fljA, fljB, flk, flmH, 6–10 ompD
lafK, motA, motB, nueA, tar/cheM, tsr 7–10 sugC

5
S. Yan et al. Microbial Pathogenesis 173 (2022) 105808

Fig. 4. The number of virulence genes in 36 serovars.

existed in Indiana, Pomona and Schwarzengrund, and inner membrane

protein coding gene avrA were only absent in Indiana.
In addition, some of the same serotypes could be further subdivided
into different ST, and we found there is a close relationship between ST
and virulence gene profiles. For instance, four strains of Newport could
be divided into two STs, ST46 (QLUY301, QLUY306) and ST166
(QLUY305, QLUY106), and two ST strains had their own virulence gene
profiles (Fig. 2).

3.7. Relationships analysis of virulence genes and sources, years or

locations of isolation

Although virulence genes were closely related to serovars, there were

slight differences of virulence genes in the same serovar Salmonella
Fig. 5. Distribution of 351 homologous virulence genes shared with other
isolates from different sources, years or locations of isolation. As shown
genera of pathogens.
in Table 4 strains belonged to three serovars were exampled. The Cor­
vallis isolates N2 and QLUY602 from the same year and location, but N2
response by means of interfering with TLR signals [23], was only present
from human had 8 different virulence genes compared with QLUY602
in 30 isolates of all Enteritidis.
from poultry. Q6 and QLUY706 of Blockley had 2 different genes due to
the discrepancy in locations of isolation, and there were 8 different
4. Discussion
genes between A2 and D2 of Enteritidis because of years of isolation.

Bacterial pathogenicity is closely related to its owned virulence

3.8. Homologous virulence genes shared with other pathogens
genes. When virulence genes expressed, their encoding products provide
survival advantages to bacteria and have negative effects on the host [9,
In the annotation results of virulence genes, some genes showed to be
24]. However, conventional low-throughput gene investigation methods
derived from other genera, which were named as “homologous viru­
for well-known virulence genes cannot simultaneously analyze complete
lence genes”. In our results, there were 351 virulence genes in Salmonella
virulence genes profiles on a large scale so as to reveal potential path­
shared with other pathogens, such as Escherichia, Haemophilus and Yer­
ogenicity. WGS is a revolutionary approach for bacterial identification,
sinia. The proportion and detailed information of virulence genes shared
genotyping, tracing, propagating, pathogenicity and phylogenetic
with other pathogens were shown in Fig. 5 and Table S4. Some of them
analysis. In this study, the virulence genes profiles of 243 Salmonella
also showed intensive relationships with serovar, such as Toll/
isolates from different sources, years, and locations in China were
interleukin-1 receptor domain protein coding gene btpA, firstly re­
investigated using WGS combined with VFDB database aligning.
ported in Brucella, with the function of inhibiting innate immune

Table 4
The relationships between virulence genes and sources, years and locations of isolation.
Serovar Strain ID Source Year Location Numbers of virulence gene Differential virulence genes

Corvallis N2 Human 2008 Henan 490 virB, pilN, pilQ, pilM, pilS, pilU, pilP, sciJ
QLUY602 Poultry 2008 Henan 482 none
Blockley Q6 Poultry 2010 Sichuan 476 Fphi_1039
QLUY706 Poultry 2010 Henan 476 trwE
Enteritidis A2 Poultry 2012 Sichuan 468 YPK_3178, pilQ, pilN, ABZJ_00085, pilV, pilU, ABZJ_00086
D2 Poultry 2010 Sichuan 462 manB

6
S. Yan et al.
A total of 670 virulence genes were obtained, and 319 virulence
genes were present in all Salmonella isolates, including 9 virulence genes
exclusively existed in Salmonella and never in other bacteria. Because
their nucleotide sequences are extremely conserved, theses 9 exclusive
virulence genes can be potentially developed as molecular markers of
Salmonella identification and pathogenesis. In addition, the virulence
genes with high detection rates underscored bacterial potential patho­
genicity so as to cause salmonellosis or public food safety crisis [25].
The function category analysis of virulence genes showed that
adhesion-related genes accounted for a large proportion, which
contribute to form biofilms and improve bacterial adhesion and colo­
nization abilities so as to enhance the pathogenicity of bacteria [26].
Previous report demonstrated that type I fimbriae was highly conserved
in Salmonella, forming hollow helical structures by non-covalent bonds
between protein subunits [27], and 80% of Salmonella species contained
the gene operons encoding type I fimbriae. In our study, fim genes
related to the type I fimbriae expression were with the detection rates of
100% in Salmonella. Some adhesion-related genes, such as csg, bcf, stb,
stf, sth operons, ratB, shdA, sinH, were also with detection rates more
than 97.12% (Table S3). Certainly, some adhesion-related genes, such as
sef, sta, tcf and misL, only existed in a portion of strains (12.35%–
56.38%) (Table S3). Previous studies [28,29] verified that shdA, ratB
and six types of fimbriae operons (i.e. lpf, bcf, stb, stc, std, sth) were
involved in intestinal tract long-term colonization of Salmonella in
genetically resistant mice. Our results showed that shdA, ratB, lpf, bcf,
stb, stc, and sth were with the detection rate more than 67%. So we
speculated that most of the tested strains had the capacity to colonize
intestine. It was proved that misL-coding protein was involved in the
interaction between host and pathogens in the process of infection,
which was conducive to colonization and long-term existence in intes­
tinal tract [12]. Therefore, misL may be a potential indicator of the
adhesion performance.
In Salmonella, there are two T3SS (T3SS-1 and T3SS-2) with a clear
division of labor in coordination and cooperation [30]. T3SS are
responsible for injecting effector proteins into host cells to alter cellular
functions, such as cytoskeleton, cell signaling cascades, and immune
factor expression, thereby promoting the invasion and proliferation of
pathogens in host as well as protecting pathogens from being eliminated
by host immune system [31]. We identified a large number of virulence
genes associated with T3SS, including prg, hil, org, inv, spa operons
encoded by SPI-1, and ssa, ssr, ssc, and sse operons encoded by SPI-2.
These genes coding proteins were the basis of the virulence factors of
Salmonella. However, these T3SS associated virulence genes respectively
present different detection rates. For example, sspH2 was only presented
in three isolates, and the detection rates of sopE, sseI/srfH, gogB were
relatively low, their frequencies ranged from 16.05% to 31.69%.
Salmonella virulence plasmid is about from 50 kb to 90 kb in size and
is closely related to its virulence [14,32]. In this study, 11
plasmid-coding virulence genes were investigated. Among them, rck
existed in all isolates while the others accounted for from 13.99% to
15.64%. spv genes are necessary for bacteria to survive, grow and
reproduce in host macrophages, and exhibit cytotoxicity to trigger
apoptosis of macrophages. Previous studies reported that some Salmo­
nella serovars, including Enteritidis, Typhimurium, Dublin and Choler­
aesuis, possess virulence plasmids [9]. In this study, we found that the
virulence genes encoded by plasmid were present in some isolates of
Enteritidis and Typhimurium, and rck even presented in all isolates. In
addition, pefB was detected in Havana, and mig-5 as well as spv existed in
one isolate of Saintpaul. Thus, virulence plasmids may be present in a
variety of serovars and can be transferred between strains, demon­
strating the pathogenic potential of various serovars strains.
The transcription activator PhoP/PhoQ is a key regulator of many
virulence genes involved in pathogenicity of Salmonella [33]. Previous
study shewed that Typhimurium with a defect of the phoP/phoQ genes
reduced virulence in mice [15]. In this study, these two genes existed in
all the tested Salmonella strains, indicating that all the tested strains had
7
Microbial Pathogenesis 173 (2022) 105808
potential pathogenicity. cdtB, encoded by SPI-3, played a role in
apoptosis and necrosis [34]. In this study, cdtB accounted for 11.52%,
and only presented in serovars Indiana, Schwarzengrund and Pomona,
showing strong correlations with serovar.
Previous studies have confirmed the important role of repetitive
sequences in bacterial genome [35], which also explain the paradox that
Salmonella strains owned similar virulence genes show different patho­
genicity [36]. We analyzed the copy number of 319 virulence genes
existed in all the Salmonella tested strains. Different virulence genes
owned different copies and several conservative virulence genes with
many copies in Salmonella were reported for the first time. Certainly, the
relationships between copy number and specific functionality need to be
verified by further investigation.
We analyzed the relationships between Salmonella virulence gene
profiles and serovars, STs, and found that different strains belonged to
the same serovar or STs have their own virulence gene profiles. This
findings were consistent with the previous study that some virulence
genes were particularly associated with Enteritidis [37]. Meanwhile,
Salmonella strains from different sources, locations and years of isola­
tion, owned different virulence gene profiles. Previous research revealed
that Salmonella adapted to new hosts and environments by gaining or
losing virulence factors [38]. In this study, the tested strains were iso­
lated from multiple sources in different regions with a long time span,
the selective pressure and accumulation of evolution in different niches
created bacterial diverse virulence gene profiles. Therefore, virulence
gene profiles analysis of Salmonella from different sources is beneficial
for molecular epidemic investigation, Salmonella monitoring, and ac­
curate prevention and control.
We also found that some homologous virulence genes shared with
other pathogens in this study. A previous study reported a large number
of homologous virulence gene were found in marine bacteria and may be
related to bacterial infection and survival [39]. Recent studies also
showed that ryhB in Escherichia coli was also present in Salmonella
enterica, Yersinia pestis, Pseudomonas aeruginosa, Vibrio cholera and
Shigella dysenteriae [40], and fyuA, irp-1 and irp-2 of the Yersinia High
Pathogenicity Island (HPI) were also present in Salmonella for the first
time [41]. Horizontal gene transfer (HGT) is a key factor that cannot be
ignored in explaining homologous virulence genes among pathogens,
which is mediated by plasmid, pathogenicity islands, integrons, trans­
posons, and other genetic elements. Reports have shown that HGT can
increase the adaptability and competitiveness of pathogens in a new
niche through the exchange of exogenous virulence genes or drug
resistance genes captured by integron within or between bacterial
genera [38,42]. These findings reflected bacterial evolutionary conser­
vation and the potential commonalities in exerting virulence.
5. Conclusion
An insight into the virulence genes of Salmonella enterica was un­
dertaken to assess the molecular epidemiological characteristics and
pathogenic potential based on WGS and VFDB analysis. We identified a
large number of virulence genes in Salmonella enterica, especially 9
unique virulence genes that can be potentially used as molecular
markers for Salmonella detection. Most of them are associated with
adherence and effector delivery systems, suggesting the potential viru­
lence of Salmonella. The heterogeneity of copy number of some con­
servative genes were also demonstrated in this study. There was a strong
correlation between virulence genes and serovars, STs. The strains
belonged to the same serovars and STs had similar virulence gene pro­
files. Meanwhile, sources, regions and years of isolation could also affect
bacterial virulence gene profiles, which might be the results of gene
selective expression in different ecological niches and period. In addi­
tion, we also found many homologous genes shared with other patho­
gens, which indicated the evolutionary conservation of virulence genes
in different genera of pathogens. In summary, our results are beneficial
for further understanding of Salmonella molecular pathogenesis,
S. Yan et al.
investigation and monitoring, as well as treatment and prevention of
salmonellosis.
Funding
This work was funded by the National Key Research and Develop­
ment Program of China (grant number 2017YFC1601404), the Natural
Science Foundation of Shandong province (grant number
ZR2018LC003) and the Key Research and Development Program of
Shandong Province (grant number 2019GNC106154, 2014GGB01661).
CRediT authorship contribution statement
Shigan Yan: Writing – review & editing, Supervision, Methodology,
Investigation, Funding acquisition. Xu Liu: Writing – original draft,
Investigation, Formal analysis. Chengyu Li: Investigation, Data cura­
tion. Zhaoxu Jiang: Investigation. Donghui Li: Investigation. Liping
Zhu: Writing – review & editing, Supervision, Investigation, Funding
acquisition.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Data availability
No data was used for the research described in the article.
Supplementary Materials
Additional file 1: Table S1 Background information and number of
virulence genes in 243 Salmonella enterica strains.
Additional file 2: Table S2 Genomic information of 243 Salmonella
enterica strains.
Additional file 3: Table S3 Detection rates of 670 virulence genes in
Salmonella strains.
Additional file 4: Table S4 Detailed information of 315 homologous
virulence genes shared with other pathogens.
(A) The number of Salmonella isolates of each serovar from different
sources. (B) The number of Salmonella isolates from different locations.
(C) The number of Salmonella isolates from different sampling years.
Because the other 319 virulence genes were present in all strains, the
heat map only showed the presence of 351 virulence genes. Each row
represents a virulence gene, and each column represents a single isolate.
The legend on the right of the figure depicts the detection of virulence
genes in the strains, with red indicating presence and white indicating
absence.
The abscissa symbolizes the detection rates of virulence genes in the
tested Salmonella isolates, every 20% as a data interval. The ordinate
represents the number of virulence genes corresponding to different
detection rates.
The abscissa represents the 36 serovars corresponding to all isolates,
and the ordinate represents the number of virulence genes of different
serovar strains.
The vertical axis represents distinct bacterial genera, and the hori­
zontal axis indicates the number of homologous virulence genes shared
with different bacterial genera. Only the genera with more than 10 genes
were listed in this figure, and information of the other genera was shown
in Table S4.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.micpath.2022.105808.
8
Microbial Pathogenesis 173 (2022) 105808
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