Distribution of genes encoding virulence factors of Shigella strains isolated from children with diarrhea in southwest Iran

This study aimed to investigate the distribution of virulence factor genes in Shigella strains isolated from children with diarrhea in the southwest, Iran. In this cross-sectional study, 1530 diarrheal stool specimens were collected from children aged under 15 years. The Shigella strains were identified by biochemical methods and polymerase chain reaction (PCR). Subsequently, all Shigella isolates were evaluated by PCR for the presence of nine virulence genes ipaH (responsible for dissemination from cell to cell), ial (responsible for epithelial cell penetration), sat (displays cytopathic activity in several intestinal cell lines), sigA (toxic to epithelial cells), pic (associated with colonization), pet (cytotoxic for epithelial cells), sepA (contribute to intestinal inflammation and colonization), virF and invE (regulatory proteins). A total of 91 isolates including 47 S. flexneri, 36 S. sonnei, and 8 S. boydii were identified. All isolates were positive for the ipaH gene. The other genes include ial, virF, invE, sigA, sat, sepA, pic and pet found in 84.6%, 72.5%, 68.1%, 62.6%, 51.6%, 39.5%, 37.3% and 28.5% of the isolates, respectively. The results showed a high distribution of virulence genes among Shigella strains in our region. It seems that for different Shigella spp. different virulence factors contribute to pathogenesis. The current study provided insights into some baseline information about the distribution of some virulence genes of Shigella isolates in Southwest Iran.


Introduction
Shigellosis is an acute gastroenteritis infection caused by Shigella species. It is one of the most common causes of morbidity and mortality, especially in children in developing countries [1]. Shigellosis is characterized by fever, abdominal cramps, mucoid stool, and bloody diarrhea [2]. The severity of Shigellosis is depended on the various virulence factors located in the chromosome or large virulent inv plasmids [3]. The invasion plasmid antigen H (ipaH) genes are present in multiple copies located on both a plasmid and the chromosome are responsible for dissemination in epithelial cells. The invasion-associated locus (ial) gene, which is located on a plasmid, is involved in cell penetration by Shigella [4]. Two regulatory proteins, virF and invE are involved in the control the transcription of invasion genes [5]. The serin autotransporters proteins of Enterobacteriaceae (SPATEs) are present in Shigella strains. The SPATEs family has been divided into two classes. Class 1 SPATEs members include the plasmid-encoded toxin gene (pet), secreted autotransporter toxin gene (sat), and Shigella IgA-like protease homolog gene (sigA), which are cytotoxic for epithelial cells. The protease involved in colonization of the intestine (pic) and the extracellular protein Shigella A (sepA) are members of class 2, which contribute to intestinal inflammation and colonization [6]. Shigella isolates harboring virulence genes can induce extensive mucosal damages and inflammation in intestinal cells, especially when these strains encode more than one of the mentioned virulence factors.
Despite many reports about the prevalence and antimicrobial resistance of Shigella from different parts of the world and Iran, investigations about the prevalence of virulence factors in Shigella spp. are still rare worldwide. Therefore, we investigated the distribution of genes encoding virulence factors of Shigella strains isolated from children with diarrhea in southwest Iran.

Bacterial isolation
In this study, 1530 stool samples were collected from patients with diarrhea referring to the teaching hospitals in Ahvaz and Abadan, southwest of Iran, for 18 months from April 2017 to September 2018. Our study included patients with a history of fever, abdominal cramps, vomiting, watery and bloody diarrhea. Patients who had taken antibiotics during the 72 h prior to the time of sampling were excluded. Diarrhea was defined as having at least three loose or liquid stools without blood and mucus per day. Dysentery was characterized by inflammation of the intestine, frequent excretion (10-13 times a day) of slimy stools that often contain blood, pus, and mucus. All isolates were identified as Shigella strain by standard microbiological and biochemical tests as previously described [7]. Pure culture of bacterial isolates was kept in a micro-tube containing Tryptic Soy Broth (TSB) (Merck, Germany) with 30% glycerol at −70 °C until molecular analysis. All Shigella strains were subsequently confirmed by polymerase chain reaction (PCR) assay [7]. Oligonucleotides and genes used for the identification of Shigella species were described in Table 1.

Molecular identification of Shigella sp.
Shigella strains were evaluated for the prevalence of rfpB, wbgZ, rfc, and hypothetical protein genes. DNA extraction of isolates was performed by the boiling method [8]. PCR amplification was performed according to the previous study [7]. The sequences of species-specific primer are listed in Table 1. Amplification reaction was carried out in Thermal Cycler Gradient (Eppendorf, Germany). The PCR conditions were: 94 °C for 5 min, 35 cycles at 94 °C for 1 min, annealing (variable) for 1 min, 72 °C for 1 min, and a final step of 72 °C for 7 min. The PCR amplification products were electrophoresed on a 1.5% agarose gel stained with ethidium bromide and visualized in the gel documentation (Protein Simple, CA, USA). We used S. sonnei (ATCC25931), S. flexneri (ATCC29903), S. boydii (ATCC8700), and S. dysenteriae (ATCC13313) as the positive controls, and PCR mixture without DNA template as the negative control.

PCR amplification of virulence genes
We performed PCR assays that targeted nine different virulence gene factors (ipaH, ial, virF, invE, pet, sat, sigA, pic, and sepA) using the primers described in Table 2. The total volume of the PCR mixture was 25 μL, containing 0.5 μL of DNA template, 1× PCR buffer, 2.5 Mm of MgCl 2 , 0.5 μL each virulence gene primer, 0.5 μL Taq DNA polymerase. The PCR conditions for the amplification of virulence genes included an initial denaturation at 94 °C for 60 s, 35 cycles of denaturation at 94 °C for 60 s, annealing (Table 2) for 60 s, and extension at 72 °C for 60 s, as well as a final extension at 72 °C for 7 min. The amplicons were separated in 1.5% agarose gel. Positive controls for each of the virulence gene were as follows: S. flexneri ATCC 12122 for ipaH, S. flexneri 2a strain 2457T for sat, sepA and sigA, enteroinvasive Escherichia coli (EIEC) strain 44825 for invE, S. flexneri ATCC 12122 for virF, enteroaggregative Escherichia coli (EAEC) strain 042 for pet and pic, EIEC strain 43893 for ial.

Statistical analysis
The descriptive statistic tests were performed in SPSS version 22. Correlation between the occurrence of virulence factor genes and multidrug resistance was calculated using Fisher's exact test. A (P value <0.05) was considered statistically significant.

Frequency of virulence factors genes
All isolates were positive for the ipaH genes (responsible for dissemination from cell to cell). The detection of the virulence genes from 91 Shigella isolates 84.6% (n = 77) of isolates were positive for ial (responsible for epithelial cell penetration), whereas 72.5% (n = 66) and 68.1% (n = 62) were positive for the virF and invE genes (regulatory proteins). The data revealed that sigA (toxic to epithelial cells), sat (displays cytopathic activity in several intestinal cell lines), sepA (contribute to intestinal inflammation and colonization), pic (associated with colonization) and pet (cytotoxic for epithelial cells) genes were present in 62.6% (n = 57), 51.6% (n = 47), 39.5% (n = 36), 37.3% (n = 34) and 28.5% (n = 26) of the isolates, respectively. All Shigella isolates harbored at least one SPATE gene. All S. flexneri isolates harbored sat gene (P < 0.05), but all the S. sonnei and S. boydii isolates were negative for this gene. The prevalence of these genes among the Shigella spp. is shown in Table 3.

Discussion
Shigellosis is an acute invasive enteric infection. It is one of the important causes of morbidity and mortality in developing countries, especially among children younger than 5 years [9,10]. In the current study, a total of 91 Shigella spp. were isolated from diarrhea specimens of children aged under 15 years in Ahvaz, Abadan, southwest Iran. The most frequent age group in our study was age 1-5 years (P < 0.05), which was consistent with previous studies [4,11]. Children in this age group due to poor personal hygiene and lack of previous exposure and lower immune responses are more prone to shigellosis [9]. Epidemiological studies have shown that the distribution of four Shigella species is varied in different geographical areas, and S. flexneri was recognized as the major bacterial causative for diarrhea in many developing countries [3]. However, S. sonnei is the most commonly isolated species in many developed countries [12,13]. In the current study, the prevalence rate of Shigellosis in diarrheal children was 5.9% and S. flexneri (51.6%) was the predominant species among Shigella species in our region, which is comparable with previous studies [3,11,14]. We also did not succeed in isolating S. dysenteriae, which was in accordance with some other studies conducted in Iran [15][16][17]. However, other researchers in our region have reported the S. dysenteriae as the less common species [18,19]. S. dysenteriae is associated mainly with outbreaks and epidemics which often found in South Asia and sub-Saharan Africa [20]. Shigella invades epithelial cells of the colon and kills them. The genes related to the invasion of Shigella are located on the chromosome and plasmids [21]. Several virulence genes associated with Shigella pathogenesis have been identified. Identifying the virulence-associated genes in Shigella strains is useful to better understand its pathogenicity. In this study, we investigated the prevalence of nine virulence genes in Shigella isolates. The ipaH gene used as a diagnostic marker for Shigella detection because this gene is found both on the chromosome and plasmids. In our study, the ipaH gene was positive for all the isolates, whereas the ial gene was detected in 84.6% that is consistent with other studies [14,22,23]. It seems that because the ial gene is only located on the Inv plasmid, it is prone to be lost or deleted. In the current study, the prevalence of the invE and virF genes was 64.8% and 69.2%, respectively. These results are consistent with a previous study [23]. Since virF and invE genes are located on the plasmid, they are susceptible to elimination.
Other genes that possess virulence activities are SPATE genes, which encode for secreted autotransporters in gramnegative bacteria. There is little information about the distribution of SPATE genes in Shigella isolates. In the present study, the SigA gene has the highest frequency among class 1 SPATE genes, which is consistent with previous studies [5,23]. Our results implied that sigA may play an important role in the pathogenesis of Shigella. In our study, the sat gene was found in 100% of S. flexneri strains. In agreement with our finding Hosseini Nave et al. and Roy et al. showed that sat were present almost in all S. flexneri strains, but it was not found in any of S. sonnei and S. boydii strains [23,24]. This gene can cause damage to the intestinal epithelial cells and therefore plays a role in pathogenesis [25].
The pic and sepA genes were detected in 39.5% and 37.3% of isolates, respectively. Our results matched with the previous study from Kerman, Iran [23]. The sepA gene is located in the virulence plasmid, and close to the pic gene located on the chromosome. Due to storage or subculturing the plasmid might have been lost together with the sepA gene. These genes can cause fluid accumulation, and to the successful colonization of Shigella isolates in intestinal cells [26]. In our study, S. boydii isolates had high rates of class 2 SPATE genes (sepA and pic) that is in agreement with the previous study [23].

Conclusions
In the present study, we provided some baseline information about the distribution of some virulence genes in clinical strains of Shigella spp. in southwest Iran. These results showed a high distribution of virulence genes among Shigella strains in our region. It seems that for different Shigella spp. different virulence factors contribute to pathogenesis. It was found that the profile of these virulence genes correlated with serotype, period, and region. consent was obtained from all the children's parents. Our manuscript has been already submitted to a pre-print platform (Research Square), providing the DOI (https ://doi.org/10.21203 /rs.3.rs-29988 /v1) and licensing information.