DOI: https://doi.org/10.21203/rs.3.rs-29988/v1
Background: This study aimed to investigate the distribution of virulence factor genes in Shigella strains isolated from children with diarrhea in the southwest, Iran.
Methods: In this cross-sectional study, a total of 91 Shigella spp. were isolated from diarrhea specimens of 1 530 children aged under 15 years in Ahvaz and Abadan, southwest Iran. The Shigella strains were identified by biochemical methods. Subsequently, Shigella spp. were identified by polymerase chain reaction (PCR).All Shigella spp evaluated by PCR for the presence of 9 virulence genes (ipaH, ial, virF, invE, sat, sigA, pic, pet, and sepA).
Results: 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.
Conclusion: Our 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.
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 a both plasmid and the chromosome is responsible for dissemination in epithelial cells [4]. 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 control the transcriptional of invasion genes [5]. The serin autotransporters of proteins 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 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.
In this cross-sectional 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. Patients with a history of fever, abdominal cramps, vomiting, watery and bloody diarrhea were included in our study. All isolates were identified by standard biochemical tests [7]. All isolates that confirmed as Shigella spp. were stored in Tryptic Soy Broth (TSB) (Merck, Germany), containing glycerol (30%) at − 70 °C.
All Shigella strains were evaluated for the presence of the ipaH gene by PCR. DNA extraction of isolates was performed by the boiling method previously described [8]. The sequences of primers and annealing temperatures of the ipaH gene are shown in Table 1. PCR conditions were examined according to the protocol as described previously [6]. S. flexnery ATCC 12122 was used as a positive PCR control for the ipaH gene.
| Target gene | Primer sequences | Product size (bp) | Annealing Temperature (°C) | References |
|---|---|---|---|---|
| ipaH | F-GAAAACCCTCCTGGTCCATCAGG R-GCCGGTCAGCCACCCTCTGAGAGTAC | 619 | 58 | 6 |
| sat | F-TCAGAAGCTCAGCGAATCATTG R-CCATTATCACCAGTAAAACGCACC | 930 | 59 | 6 |
| ial | F- CTGGATGGTATGGTGAGG R- GGAGGCCAACAATTATTTCC | 320 | 59 | 6 |
| virF | F- TCAGGCAATGAAACTTTGAC R- GGGCTTGATATTCCGATAAGTC | 618 | 58 | 6 |
| invE | F-CGATAGATGGCGAGAAATTATATCCCG R-CGATCAAGAATCCCTAACAGAAGAATCA | 766 | 59 | 6 |
| sigA | F- CCGACTTCTCACTTTCTCCCG R- CCATCCAGCTGCATAGTGTTTG | 430 | 58 | 6 |
| pet | F-GGC ACA GAA TAA AGG GGT GTT T; R-CCT CTT GTT TCC ACG ACA TAC | 302 | 58 | 9 |
| pic | F- ACTGGATCTTAAGGCTCAGGAT R- GACTTAATGTCACTGTTCAGCG | 572 | 58 | 6 |
| Sep A | F- GCAGTGGAAATATGATGCGGC R- TTGTTCAGATCGGAGAAGAACG | 794 | 58 | 6 |
PCR was carried out on all Shigella strains to evaluate the prevalence of the rfc, wbgZ, rfpB, hypothetical protein genes. PCR was performed according to a previous study [7].
The specific primers and annealing temperatures of Shigella spp. genes are listed in Table 2. S. sonnei ATCC25931, S. flexneri ATCC29903, S. boydii ATCC8700, and S. dysenteriae ATCC13313 were used as a positive control.
| Target gene | Primer sequences | Product size (bp) | Annealing Temperature (°C) | References |
|---|---|---|---|---|
| Hypothetical protein | F: GAGCACGGAAACAGAGAGCGCC R: GGTGCGTTCTTCCGGTGTTCTG | 240 | 63 | 7 |
| wbgZ | F: TCTGAATATGCCCTCTAC R: GACAGAGCCCGAAGAACCG | 430 | 60 | 7 |
| Rfc | F: TTTATGGCTTCTTTGTCG R: CTGCGTGATCCGACCATG | 537 | 60 | 7 |
| rfpB | F: TCTCAATAATAGGGAACACAGC R: CATAAATCACCAGCAAGGTT | 211 | 59 | 7 |
PCR was performed for all Shigella strains to evaluate the prevalence of the ial, virF, invE, pet, sat, sigA, pic, and sepA [6, 9]. The sequences of primers and annealing temperatures are shown in Table 1. The total volume of the PCR mixture was 25 µL, containing 0.5 µL of DNA template, 1X PCR buffer, 2.5 Mm of MgCl2, 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 seconds, 35 cycles of denaturation at 94 °C for 60 seconds, annealing (variable) for 60 seconds, and extension at 72 °C for 60 seconds, as well as a final extension at 72 °C for 7 min. After performing PCR, the size of each locus was easily determined on 1.5% gel agarose. Positive controls for each gene were as follows: S. flexnery ATCC 12122 for virF, EIEC strain 44825 for invE, EIEC strain 43893 for ial, S. flexneri 2a strain 2457T is for sat, sepA and sigA, EAEC strain 042 is the positive control for pet and pic.
The descriptive statistic tests were performed in SPSS version 16.00. 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.
In this study, 5.9% (n = 91) of 1 530 stool samples were positive for Shigella spp. Of the 1 530 patients, 47.1% (n = 720) and 52.9% (n = 810) were males and females, respectively. The patients have had various clinical symptoms, including vomiting 31.5%) n = 482), fever 60.9%) n = 932), abdominal pain 83.1% )n = 1 271), watery diarrhea 77.9%) n = 1 193), and dysentery 21.2%) n = 324).
From a total of 91 Shigella spp., 56.0% (n = 51) and 44.0% (n = 40) were isolated from male and female patients, respectively. No significant differences in Shigella infection were found between male and female patient (P > 0.05). Distribution of Shigella spp. isolated from the 91 diarrheic children according to age were: 1–5 years, 59.3% (n = 54); 6–10 years, 24.1% (n = 22); 11–15 years, 16.5% (n = 15). Bloody diarrhea, mucoid diarrhea and watery diarrhea were found in 14.3% (n = 13), 7.7% (n = 7), 62.6% (n = 57) patients, respectively. Of these 91 positive samples, 51.6% (n = 47), 39.6% (n = 36) and 8.8% (n = 8) samples were identified as S. flexneri, S. sonnei, and S. boydii respectively. Distribution of Shigella strains according to age group and species are shown in Table 3.
| Age group, year | S. flexneri | S. sonnei | S. boydii |
|---|---|---|---|
| 1–5 | 26 (28.6%) | 22 (24.2%) | 6 (6.5%) |
| 6–10 | 13 (27.6%) | 7 (19.4%) | 2 (2.1%) |
| 11–15 | 8 (17.02%) | 7 (19.4%) | 0 (0%) |
| Total | 47 (51.6%) | 36(39.6%) | 8(8.8%) |
All isolates were positive for the ipaH genes. The detection of the virulence genes from 91 Shigella isolates 84.6% (n = 77) of isolates were positive for ial, whereas 72.5% (n = 66) and 68.1% (n = 62) were positive for the virF and invE genes. The data revealed that sigA, sat, sepA, pic and pet genes found 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 4.
|
virulence genes S. flexneri S. sonnei S. boydii Total |
|
pic 18(38.2%) 10(27.7%) 6(75%) 34(37.3%) |
|
sepA 18(38.2%) 12(33.3%) 6(75%) 36(39.5%) |
|
pet 11(23.4%) 15(41.6%) 0% 26(28.5%) |
|
sigA 31(65.9%) 19(52.7%) 7(87.5%) 57(62.6%) |
|
sat 47 (100%) 0 ( 0% ) 0 (0%) 47 (51.6%) |
|
ial 38(80.8%) 31(86.1%) 8(100%) 77(84.6%) |
|
virF 26(55.3%) 29(80.05%) 7(87.5%) 62(68.1%) |
|
invE 28(59.9%) 31(86.1%) 7(87.5%) 66(72.5%) |
Distribution of the virulence genes according to Shigella species.
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 [10, 11]. 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, 12]. Children in this age group due to poor poor personal hygiene and lack of previous exposure and lower immune responses are more prone to shigellosis [10]. Shigella invades epithelial cells of the colon and kill them. The genes related to the invasion of Shigella are located on chromosome and plasmid [13]. Several virulence genes associated with Shigella pathogenesis have been identified. Identifying the virulence-associated genes in Shigella strains would be useful to better understand its pathogenicity. In this study, we investigated the prevalence of 9 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 the plasmid. 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 studies that agree with other studies [14–16]. It seems that because of the ial gene is only located on the plasmid, it is prone to lose or deletion. In the current study, the prevalence of the invE and virF genes was 64.8% and 69.2%, respectively. These results are consistent with the previous study [16]. 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 secreted autotransporters in gram-negative 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 [5,16 ]. 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 [16, 17]. This gene can cause damage to the intestinal epithelial cells and therefore plays a role in pathogenesis [9].
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 [16]. The sepA gene located on the virulence plasmid, and by 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 lade to fluid accumulation, the successful colonization of Shigella isolates in intestinal cells [18]. 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 [16].
In the present study, we provided some baseline information about the distribution of some virulence genes in clinical strains of Shigella spp. Southwest Iran. Our 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.
The study was approved by the Research Ethics Committee of the Abadan School of medical sciences (Ethical code: IR.ABADANUMS.REC1398.073), Abadan, Iran. Written informed consent was obtained from all the children’s parents.
This work was supported by the Vice-Chancellor for Research grant (Grant No.98U-603) of Abadan University of Medical Science. The authors of this manuscript would like to acknowledge the laboratory and nursing personnel of children and infants ward in teaching hospitals in Ahvaz, Abadan and khorramshahr, who assisted to collect the clinical specimens.
Maryam Afzali developed the original idea and the protocol, performed the experiments, Khadijeh Ahmadi was involved in data collection, wrote the preliminary draft and analyzed the data, Nabi Jomehzadeh revised the manuscript.
The authors report no conflicts of interest in this work.
All data generated or analysed during this study are included in this published article.