Investigating the Prevalence of Shigella Species and Their Antibiotic Resistance Pattern in Children With Acute Diarrhea Referred to Selected Hospitals in Tehran , Iran

Caused by Shigella species, shigellosis is an infectious disease which is endemic in many developing countries.1 Annually, there are about 80 million cases of bloody diarrhea and 700 000 deaths due to this infection.2 Children under 5 years old, elderly, and immunocompromised people are at a higher risk to this infection and develop more severe symptoms.3,4 The majority of the disease cases are seen in developing countries and it is due to the poor hygiene standards, poor water quality, etc.5 Four different species of Shigella genus can develop the disease, Shigella sonnei, Shigella dysenteriae, Shigella flexneri, and Shigella boydii. The incidence of the species is so different in various countries: while S. flexneri is the prevalence serogroup in developing countries, the most cases of the infection in developed countries are caused by S. sonnei.6,7 Since it is transmitted via fecal-oral rout in a low dose (1-500 organisms) and also via direct spread by person to person contact, the control of the infection is a great challenge.8,9 Shigella infection can cause different symptoms from a mild self-limiting diarrhea to severe dysentery associated with blood and mucus excretion, high fever, and cramps.10,11 Since there is no approved vaccine, despite all attempts,12-14 antibiotic therapy is the main strategy to combat the disease. Appropriate antibiotic therapy shortens the disease symptoms and prevents the life-threatening effects, also, it decreases the bacterial shedding in feces and, therefore, decreases the bacterial spread.15 However, during recent decades, the indiscriminate use of antibiotics has led to the resistance of Shigella spp. to common antibiotics.16,17 Due to the self-limiting nature of the Investigating the Prevalence of Shigella Species and Their Antibiotic Resistance Pattern in Children With Acute Diarrhea Referred to Selected Hospitals in Tehran, Iran


Introduction
Caused by Shigella species, shigellosis is an infectious disease which is endemic in many developing countries. 1Annually, there are about 80 million cases of bloody diarrhea and 700 000 deaths due to this infection. 2Children under 5 years old, elderly, and immunocompromised people are at a higher risk to this infection and develop more severe symptoms. 3,4he majority of the disease cases are seen in developing countries and it is due to the poor hygiene standards, poor water quality, etc. 5 Four different species of Shigella genus can develop the disease, Shigella sonnei, Shigella dysenteriae, Shigella flexneri, and Shigella boydii.The incidence of the species is so different in various countries: while S. flexneri is the prevalence serogroup in developing countries, the most cases of the infection in developed countries are caused by S. sonnei. 6,7ince it is transmitted via fecal-oral rout in a low dose (1-500 organisms) and also via direct spread by person to person contact, the control of the infection is a great challenge. 8,9higella infection can cause different symptoms from a mild self-limiting diarrhea to severe dysentery associated with blood and mucus excretion, high fever, and cramps. 10,11Since there is no approved vaccine, despite all attempts, [12][13][14] antibiotic therapy is the main strategy to combat the disease.Appropriate antibiotic therapy shortens the disease symptoms and prevents the life-threatening effects, also, it decreases the bacterial shedding in feces and, therefore, decreases the bacterial spread. 15However, during recent decades, the indiscriminate use of antibiotics has led to the resistance of Shigella spp. to common antibiotics. 16,17Due to the self-limiting nature of the infection, the high risk of the emergence of drug resistance strains, and the costs of drugs, some physicians avoid the antibiotic administration. 18However, there is a convincingly logic in favor of empirical treatment of shigellosis in suspected patients.If the disease won't be treated, a child may be quite sick for several weeks or more and chronic diarrhea may be created.During prolonged illness, the risks for developing or worsening the disease, malnutrition, permanent disposal of microorganisms, and subsequent infection of family members will be increased.However, the resistance of the bacteria to common antibiotics has limited the use of these antimicrobial agents.Although it was thought that Shigella is sensitive to ceftriaxone, azithromycin, and quinolones, there are increasing number of reports which show the increase in multiple drug-resistancy of the bacterial strains in different parts of the world.In Iran, more than 90% of Shigella species are resistant to one or more antibiotics and about 87% of them are resistant to more than one antibiotic. 19Since shigellosis is the worldwide health problem and it is endemic in some regions of Iran, 20 and also because of the importance of the determination of antibiotic resistant pattern of the bacteria in the selection of the proper treatment regimen, in the present study, the prevalence of Shigella species and also the antibiotic resistance pattern of them were investigated.

Materials and Methods
This is a hospital-based descriptive-analytic study based on stool samples of patients with diarrhea and fever diagnosed with colitis in four educational hospitals.

Sample Collection
Three hundred diarrhea samples were collected from four hospitals in Tehran, including Sina, Shariati, Imam Khomeini, and children medical center hospitals in a time period from June to November 2016.Sampling performed before antibiotic therapy and stool specimens were transported in Cary-Blair transport medium and carried to the microbiology laboratory for future analysis.

Statistical Analysis
The data in each experiment was a representative of three independent experiments.Statistical analysis was carried out using the SPSS software (version 24.0).

Ethical Considerations
Ethical approval was obtained from the research ethics committee of selected hospitals.

Bacterial Identification
Isolates which pose the following biochemical characteristics were considered Shigella: lactose negative, gas production negative, non-motile, MR positive, lysine decarboxylation negative, citrate negative, and finally, urea hydrolysis negative.According to these tests, out of 300 samples, 26 samples (8.7%) were detected as Shigella.For species discrimination, serological typing through specific antisera against isolates was performed.Based on serotyping, it was shown that out of 26 isolates, 15 isolates (57.7%) belonged to S. sonnei, and 11 isolates (42.3%) belonged to S. flexneri (Figure 1).

Antibiogram Test
As shown in Table 1, the result of Bauer-Kirby method demonstrated that most of Shigella isolates were resistant to tetracycline (88.46%), followed by ampicillin, chloramphenicol, and Sulfamethoxazole/trimethoprim (61.53%), while 92.30% and 85% of the isolates were sensitive or intermediate to ciprofloxacin and gentamicin, respectively.

Discussion
Shigellosis is a worldwide health problem, especially in developing countries.It is estimated that the annual morbidity rate of the infection is 80 million, which most of cases are children under 5 years old. 2 Etiologically, Shigella species are the leading cause of acute diarrhea among hospitalized children (27% of all cases). 21Different studies have shown that the prevalence of the diarrhea committed by Shigella is almost high in Iran.However, geographically the prevalence is so variable.For example, the bacterial diarrhea caused by Shigella in different cities has been as follow: Zanjan 19.6 %, 22 Ahvaz 14.1 %, 23 Kerman 31.6%, 12 Shahr-e-Kord 7.8%, 24 and Kashan 7.8%. 25In Tehran, in a study by Afshari et al in summer and autumn of 2012, it was shown that 5.3% of diarrheal cases are caused by Shigella, 21 and Dolatshahi et al, in another study in a period of 10 months from April to January 2015, showed that this rate was 5.16%. 22In the present study the prevalence of the Shigella in bacterial diarrhea cases was 8.7%, which the differences are not statistically significant.The prevalence of Shigella species pattern is different among developing and developed countries, while S. sonnei is the most common species in developed countries, the predominant Shigella species in developing countries is S. flexneri.The present study shows that the most prevalent Shigella strains in Tehran are belonged to S. sonnei and it is in agreement with Rahbar et al, 26 Talebreza et al, 27 Jafari et al, 28 Tajbakhsh et al, 29 Ranjbar et al, 30 Eftekhari et al, 31 and Farshad et al 32 reports.It is while in the not-too-distant past, S. flexneri was the most common species in Tehran 33,34 (P<0.05), which indicates the improvement of hygiene level in this city.Antibiotic therapy is the choice strategy for the treatment of patient with acute Shigella infection. 35It can reduce the symptoms of disease, decrease the number of carriers, and prevent the spread of the infection.However, the irregular use of antibiotics has arisen antibiotic resistance of the bacteria and, therefore, the treatment of the infection has been complicated. 15,32Beta lactams, quinolones, and macrolides are tree classes of antibiotics which are recommended by World Health Organization (WHO) to treat the disease.][38] In the present study, the highest resistance to antibiotics was observed for tetracycline, ampicillin, and chloramphenicol, while the most sensitivity was observed for gentamicin and ciprofloxacin.In a study conducted by Hossein-Pour et al, the highest antibiotic resistance to ampicillin was reported to be 89.6 percent and all cases were reported sensitive to the third generation cephalosporins and nalidixic acid, while the sensitivity to gentamicin, amikacin, and chloramphenicol was 89.8%, 82.5%, and 69%, respectively. 35lso in a study by Jamshidi et al, the highest resistance was observed for ampicillin and the highest sensitivity was seen to ciprofloxacin. 22In a study by Saadati et al., the prevalence of nalidixic acid resistant strains in Kerman was reported 9.7%. 18yazi et al, in Qazvin 36 and Kurdi Daryan et al, 37 in Isfahan reported that the prevalence of Nalidixic Acid resistant Shigella were 2% and 4%, respectively.In a survey by Rahbar et al, it was observed that the most strains were resistant to ampicillin and trimethoprime/sulphamethoxazole (88.5% and 98%, respectively), whereas resistance to Chloramphenicol and Ciprofloxacin was observed to be 2.5% and 1% respectively. 26he results of the present study revealed that the prevalence of nalidixic acid-resistant Shigella strains has increased to 26.82%.0][41] Various reasons can lead to the dissemination of Shigella MDR strains, including easy access to antibiotics and their irregular use. 42,43Increasing Shigella resistance to antibiotics is a serious warning that should be prevented by implementing necessary measures.

Conclusions
Shigella's common species pattern has changed from S. flexneri to S. sonnei during the recent years in Tehran.The results of this study show that the antibiotic resistance pattern among these species is also changing, therefore, regular examination of the prevalence of different species and their drug resistance pattern is necessary to make the right decisions of the proper treatment.Indeed, increasing Shigella resistance to antibiotics is a serious warning that should be prevented by implementing necessary measures.

Figure 1 .
Figure 1.The Prevalence of Shigella Species Isolated From the Studied Samples.

Table 1 .
Antimicrobial Susceptibility of Shigella Strains Isolated From Children