Background
Shigellosis is major health burden in many parts of the world. It is an acute invasive enteric infection caused by four members of
Shigella species (
S. dysenteriae,
S. flexneri,
S. boydii, and
S. sonnei). Different serotypes for these species exist including more than 30 serotypes of
S. flexneri which are categorized based on their O antigens, [
1]. Although the role of shigellosis in contributing to childhood mortality has been decreased significantly over the past few years, there are still about 28,000 children younger than 5 years of age who died of shigellosis every year [
2]. In a systematic review, [
3] it was reported that due to low economic conditions and large population density in Asian countries, over 125 million
Shigella-related infections led to 14,000 deaths per year. There are some factors contributing to the high prevalence of human
Shigella infection. One reason for the high infection rate in some developing countries is the low sanitary conditions, knowing that
Shigella spp. is transmitted via the fecal–oral route. Another important factor is that
S. flexneri possess protective mechanisms that help it to survive even at high levels of acid in the stomach, which makes it highly infectious with only 10–100 microorganisms required to cause a disease [
4].
In children, main symptoms of shigellosis vary from mild to severe which include: diarrhoea characterized by presence of blood in stool, abdominal cramping, fever, among other gastrointestinal complications. Its clinical phenotypes are determined by different virulence genes and the activity of immune system of the host. Among the many
Shigella spp.—associated virulence factors, invasion plasmid antigen (ipa) B, C, D, and H as well as invasion-associated locus (ial) facilitates its penetration into intestinal cells [
4]. As with gram-negative bacteria, these genes are important for S.
flexneri because they are components of the type III secretion system (T3SS) which is important for
S. flexneri and other gram-negative pathogenic or symbiotic bacteria in manipulating the host cell processes and establish a successful infection [
5].
Shigella enterotoxin 1 (ShET-1),
Shigella enterotoxin 2 (ShET-2) and shiga toxin (stx) are among virulence genes encoding
Shigella enterotoxins. A group of genes mostly found in
S. flexneri serotype 2 clinical samples encode ShET1, a 55 kDa protein complex [
6,
7]. ShET2 has been reported in different species of
Shigella [
7]. The stx is produced exclusively by
S. dysenteriae 1, but this species is rare in China [
8]. The transcription of invasion-related genes is controlled by two proteins, virF and virB (InvE) which are derived from plasmids [
9]. Finally,
Shigella spp. harbors toxic factors like serine protease autotransporters of enterobacteriaceae (SPATE) of which there are two phylogenetic classes [
10].
Shigella IgA-like protease homologue (sigA) and secreted autotransporter toxin (sat) belong to class 1 which are toxic to epithelial cells, while non-toxic SPATE class 2 toxins includes sepA, which facilitates intestinal inflammation and pic, a mucinase associated with colonization.
Although some studies reported the prevalence and distribution of
S. flexneri virulence genes in some regions in China, investigations to dozen virulence genes of
Shigella spp. mentioned above are still rare throughout the world, and to the best of our knowledge there is no report in China. To develop effective control strategies, it is important to conduct an epidemic study about
Shigella in terms of its drug resistance and genetic features [
11]. For this reason, we sought to explore the distribution profile and prevalence of 12
Shigella-related virulence genes obtained from patients with diarrhea in Jiangsu Province of China, and discussed the genetic diversity and clinical applications of these genes.
Discussion
Due to inadequate supply of quality water and low hygienic conditions in less developed countries,
Shigella—a cause of inflammatory diarrhea and dysentery, poses major challenges to public health sectors.
S. flexneri was the most common of the four species in many developing countries [
19,
20]. However, in developed countries,
S. sonnei is the commonest
Shigella species isolated [
21,
22]. The reason for this difference is unclear, however, it is apparent that efforts to boost sanitation and local hygiene have greatly decreased the prevalence of shigellosis and even changed the pattern in which
Shigella species are most distributed. Jiangsu Province is located in the eastern part of China, with a population about 80 million. Epidemiological analysis of
Shigella will be beneficial to the prevention and control of the infectious diseases in the region. The results of analysis of the distribution characteristics of
S. flexneri in Jiangsu Province in the present study showed that
S. flexneri 2a was the most common of the eleven serotype, which is different from the study conducted in Beijing in China reporting that
S. flexneri 4c was the most prevalent serotype among 19 serotypes [
23]. In Jiangsu Province, serotype 4c accounted for only 3.3%. However, our results matched the findings in developing countries [
19,
24] and Zhejiang Province of China [
25]. Even in Jiangsu Province, there were also differences between the various cities (Additional file
2). For example, most prevalent serotypes in Nanjing are serotypes 2b, serotypes 1b in Zhenjiang and serotypes 1a in Taizhou. What’s more, some rare serotypes were detected at specific times in specific cities, such as serotype 6 was only separated from Nanjing in 2010. High heterogeneity with regard to temporal distribution was noted in
Shigella species and serotypes, which further suggested the need for serotype-level identification to enhance the effectiveness of control strategies.
Since the information on the variety of
Shigella virulence genes in China is limited, to fully understand its pathogenicity, further research is required to advance the search for virulence-related genes for
Shigella. In the present study, the prevalence and distribution of 12 such genes was examined. In the present study,
ipaH gene was highly conserved in various serotypes. Similar findings have been shown in many other studies [
25]. The presence of many copies of this gene i.e. seven in chromosomes and five in plasmids may explain why the gene tested positive in all strains. Considering that this gene can be detected even after the loss of plasmid, it is promising target for diagnostic purposes. In
Shigella, the ability to enter host cells depends on the availability of type III-secretion-system (T3SS) which are encoded by large virulence plasmids [
26,
27].
ial gene has been identified in invasion processes and on inv plasmid [
28]. Many proteins form part of the T3SS complex which includes a needle-shaped oligomer that connects the inner and outer membrane of the bacteria. The oligomer contains invasive plasmid antigens ipaB, ipaC, and ipaD at its tip end [
26‐
29], which can be identified using upstream region of ipaB, acting as marker. The effects of deleting
ial and
ipaBCD on invasiveness of S.
flexneri are not known. Numerous studies have shown that there is a link between the ability of the
Shigella spp. strains to cause diarrhea and the presence of invasive genes in the bacteria. Mokhtari et al. [
30] showed that, unlike in asymptomatic patients, isolates from stools of patients with diarrhea contained invasive genes,
ial and/or
ipaBCD. A study by Phantouamath et al. [
31], showed that ial gene was found only in isolates from cases. In our study, 47.0%
S. flexneri’ isolates were positive for
ial gene, and 50.5%
S. flexneri’ isolates were positive for
ipaBCD gene. Comparison with other similar studies, 78.9%
S. flexneri’ isolates were positive for
ial gene in Iran [
32], and even 100% in Zhejiang of China [
25]. For the
ipaBCD gene, our result is similar to that of a study in Peru (49%) [
19], but lower than that of a study in Brazil (100%) [
33]. in this sense, the invasive ability of
S. flexneri in Jiangsu Province is not strong compared with other areas. Moreover, prevalence of virulence genes showed obvious serotype characteristics, such as none
S. flexneri 1b expressed both
ial and
ipaBCD strains. But it should be noted that the pathogenicity of
S. flexneri is also related to both the number of infected bacteria and the immunity of infected people.
Expression of
Shigella virulence genes is regulated by heat-stable nucleoid structural protein (H-NS) which downregulates their transcription during unfavorable conditions for invasion. In response to favorable environmental signals, transcription of a series of genes is activated starting from AraC-like protein gene virF, which subsequently turns on transcription of
virB regulatory genes. Thereafter, virB protein reverses the H-NS-induced inhibition on transcription which eventually turns on the virulence genes on the plasmid [
9,
34].In the present study, both
virF and
virB were found in 45.0%
S. flexneri isolates, indicating that there might be other pathways for regulating gene expression. In addition,
virF but not
virB was found in 19.8%
S. flexneri isolates, suggesting that
virF regulated virulence genes not only through
virB pathway. Interestingly, of the 545
S. flexneri, 11 strains had only
virB, which may be due to loss of the
virF gene. On the other hand, because of the importance of
virF in regulating virulence genes, potential novel antibiotics targeting
virF have gained increasing attention [
35,
36]. However, only 64.8% of the positive rate of this gene might limit this antibiotics application.
Two new enterotoxins have recently been described in
S. flexneri. One is called
Shigella enterotoxin 1 (ShET-1), which is encoded in the
set1 chromosomal gene. It has been suggested that in its active form, the ShET-1 toxin is composed of a subunit A (encoded by set1A) and five B subunits (encoded by set1B) [
37]. Other is plasmid-encoded ShET-2 (encoded by sen). ShET-1 and ShET-2 could alter electrolyte and water transport in the small intestine [
28], which is closely related to the symptoms of dehydration in the shigellosis. Prior studies reported that
set1 genes were only detected in S.
flexneri serotype 2 (2a and 2b) isolates and less so in other serotypes. In contrast, in the current study, many
S. flexneri serotypes tested positive for
set1 genes [
7,
12,
38]. In some serotypes, however, the prevalence of
set1 (
set1A and/or
set1B) was significantly lower than in other serotypes, such as
S. flexneri 1b,
S. flexneri 3b (Table
3). And interestingly, 14.9% of
S. flexneri had only one subunit of ShET-1, the question about whether a single subunit would affect the pathogenicity of ShET-1 remains to be answered, but which needs further study for verification. The association remains to be further studied.
sen gene was found in 11 serotypes, with a majority between 40 and 80%, but the serotype 1b positive rate was only 14%. The low positive rate of ShET-1 and ShET-2 in
S. flexneri 1b means that this serotype has a low ability to cause dehydration.
Another factor that possess virulence activities is the Serine protease autotransporters of Enterobacteriaceae (SPATEs), which are toxins secreted from gram-negative bacteria. Nevertheless, only a few studies have searched for the presence of their encoding genes in large
Shigella collections. A similar study in Iran found that the sat gene was present in all
S. flexneri isolates, and the presence of
sigA,
pic and
sepA genes simultaneously were existed in 35.5% of
S. flexneri [
32]. Comparing the similar study, unsurprising, the most common SPATEs among
Shigella was
sat in our study, but the positive rate of the other three genes of SPATEs was significantly higher than that of Iran. Interestingly,
sat is now recognized as a pathogenic
E.
coli, although it was initially studied in uropathogenic
E. coli strains. In comparison with previous studies on the frequencies of
sat gene in
E. coli [
39,
40], however, the presence of
sat gene in
Shigella was found to be higher. It should be noted that except for
sat gene, SPATEs of serotype 1b was significantly less than that of the other serotypes.
The virulence gene can be used to identify
Shigella, which had been confirmed by previous studies. Some studies [
41,
42] reported that the positive rate of detecting
Shigella by a PCR assay targeting the
ipaH gene was higher than that by the traditional culture method. The disadvantage of this method is that it can only identify one virulence gene at a time, though this disadvantage could probably be overcome by multiple PCR techniques by screening the amplified genes in view of the difficulty of multiple PCR and the restriction of the number of amplified genes.
IpaH can be used as a marker gene of
Shigella to detect the
Shigella. Four genes (
pic,
set1A,
set1B and
sigA) are located on the chromosome SHI-1 Island, and the
pic gene overlaps with
set1A and
set1B. When
Shigella flexneri set1A gene was positive for
Shigella flexneri, 94.1%
Shigella set1B was positive, and 92.4%
Shigella isolates were positive for
pic and
sigA.
set1A positive
Shigella had a stronger representation of the integrity of this segment of the gene. Because of the high expression of
sat in
Shigella, the clinical value of its amplification is not significant. Other virulence genes include
ial,
ipaBCD,
virF,
virB,
sen and
sepA, all of which are located on the large virulence plasmid (140 MDa). To reflect these virulence genes of
Shigella, we chose the lowest existent
ial gene as a marker and found that the positive rate of
ial positive
S. flexneri,
ipaBCD was 98.8%, the positive rate of
virF was 96.1%, the positive rate of
virB was 92.6%, and the positive rate of
sen and
sepA was 94.5%. To sum up, multiplex PCR combination
ipaH +
set1A +
ial can comprehensively reflect the virulence of
Shigella.