Antimicrobial resistance patterns and prevalence of integrons in Shigella species isolated from children with diarrhea in southwest Iran

Nabi Jomehzadeh, Maryam Afzali, Khadijeh Ahmadi,✉, Shokrollah Salmanzadeh, Fateme Jahangiri Mehr

1Abadan Faculty of Medical Sciences, Abadan, Iran

2Infectious and Tropical Diseases Research Center, Health Research Institue, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran

3Biostatistics and Epidemiology Department, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran

ABSTRACT

KEYWORDS: Integrons; Shigella spp.; Multi-drug resistance;PCR

1. Introduction

Shigellosis is a major health-care concern in the world, especially in developing countries with poor hygiene particularly among children under 5 years old. The incidence of this infection in developing countries, to be 163 million annually[1,2]. The most common symptoms of shigellosis are vomiting, fever, watery diarrhea, tenesmus, and abdominal pain[1,2]. Shigella spp. are classified by four serogroups, including Shigella (S.) flexneri, S.boydii, S. dysenteriae, and S. sonnei. S. sonnei and S. flexneri are the most commonly found in developing countries, such as Iran[3,4].Treatment with antibiotics can reduce the duration of shigellosis but, resistance to antibiotics has been increasing. In the last decades,multidrug-resistance (MDR) has increased among Shigella spp.MDR phenotype achieves by many different mechanisms in clinical isolates. One of the important mechanisms for the increase of resistance to antibiotics is the horizontal transmission of genetic factors. Integrons are mobile genetic elements that could lead to the spread of the MDR phenotype[5]. Integron classⅠ (int

Ⅰ

)and integron classⅡ (int

Ⅱ

) are the most prevalent genes among the Shigella spp. and the relationship between the presence of integrons and resistance to some antibiotics has been demonstrated. Integrons are frequently associated with the resistance of Shigella spp. to sulfamethoxazole, trimethoprim, streptomycin, chloramphenicol,tetracycline, and ampicillin[5,6]. Although integrons play an important role in the presence of MDR in Shigella spp. there are not any data available to describe the prevalence of integrons of Shigella strains in southwest Iran, therefore, this study aimed to investigate the antimicrobial resistance patterns and prevalence of integrons in Shigella species isolated from children with diarrheal infection in the southwest of Iran.

2. Materials and methods

2.1. Bacterial isolates

In this cross-sectional study, during 18 months from April 2017 to September 2018, 1 530 stool samples were collected from children under 15 years with diarrhea referred to teaching hospitals in Ahvaz and Abadan, southwest Iran. Patients with a history of fever, vomiting, abdominal cramps, watery diarrhea and dysentery were included in our study. Dysentery was characterized by frequent excretion (usually 10 to 13 times/day) of small volume stools consisting of blood, mucus, and pus; often accompanied by abdominal cramps and tenesmus. Diarrhea was defined as the excretion of 3 or more watery stools without blood and mucus in a 24 h period. Patients who were treated with antibiotics at the time of sampling were excluded. These specimens were inoculated into Gram-negative broth tubes as an enrichment medium and immediately transferred to the Laboratory of Microbiology Department of Medicine School of Ahvaz, Iran.

All specimens were cultured in differential media, including xylose lysine desoxycholate (XLD) agar and Hektoen enteric agar (HEA)(Merck, Germany), and then incubated at 37 ℃ overnight. All grown suspected colonies were selected and identified by the biochemical and bacteriological tests such as Triple-sugar Iron Agar (TSI),Sulfide-indole-motility (SIM), Urea Agar, and Simmons Citrate Agar (Merck, Germany) for detection of Shigella strains[7]. All isolates confirmed as Shigella spp. were stored in Tryptic Soy Broth(TSB) (Merck, Germany), containing glycerol (30%) at -70 ℃ for antimicrobial susceptibility testing and molecular investigation.

2.2. Antimicrobial susceptibility

Antimicrobial susceptibility was performed on all Shigella spp. by Kirby-Bauer disc diffusion method on Muller-Hinton agar medium(Merck, Germany), according to the guidelines of the Clinical and Laboratory Standards Institute[8]. The antibiotic included ceftriaxone(30 µg), trimethoprim/sulfamethoxazole (1.25/23.75 µg), amikacin (30 µg),gentamycin (10 µg), ceftazidime (30 µg), cefotaxime (30 µg), ciprofloxacin(5 µg), azithromycin (15 µg), and ampicillin (10 µg) (Mast Ltd., UK.).Also, E. coli ATCC 25922 was used as the control strain. The phenotype of Shigella spp. was defined as MDR according to the International Expert proposal for Interim Standards Guidelines[9].The minimum inhibitory concentrations (MICs) for ceftriaxone,ceftazidime, cefotaxime, ciprofloxacin, amikacin, and gentamicin were determined by E-test (AB Biodisk, Sweden).

2.2. Molecular confirmation of Shigella strains

The whole-genome DNA was extracted using the boiling method as described in previous study[10]. All Shigella isolates were confirmed by the PCR method. PCR amplification was performed to detect the ipaH gene in Shigella isolates. 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[11]. S. flexnery ATCC 12122 was used as a positive PCR control for the ipaH gene.

2.3. PCR assay for molecular identification of Shigella species

PCR was carried out on all Shigella strains to evaluate the prevalence of the Shigella species. The primers used to detect rfc,wbgZ, rfpB, and hypothetical protein genes were as previously described[12,13]. The specific primers and annealing temperatures ofShigella spp. genes are listed in Table 1. The total volume of PCR reaction was 25 µL prepared as follows: 12.5 µL of 2X Master Mix, 1 µL of each primer (Cinna gene Company, Iran), 1 µL of template DNA, and distilled water to reach a total volume of 25 μL. Amplification reaction was programmed by a thermal cycler(Eppendorf, Germany) as follows: initial denaturation at 94 ℃ for 5 min, 35 cycles of 94 ℃ for 60 s, annealing (Table 1) for 90 s,extension 72 ℃ for 1 min and final extension 72 ℃ for 7 min. S.flexneri ATCC29903, S. sonnei ATCC25931, S. boydii ATCC8700,and S. dysenteriae ATCC13313 were used as a positive control.

Table 1. Primers used in this study to detect Shigella spp. and integrons genes.

2.4. Amplification of integrons genes

PCR was performed for the detection of int

Ⅰ

, int

Ⅱ

and int

Ⅲ

genes. The PCR conditions were similar to the previous study[14].The sequences of primers and annealing temperatures are shown in Table 1. S. flexneri ATCC 12022, S. sonnei ATCC 9290 were used as a positive control and E. coli ATCC 25922 was used as the negative control.

2.5. Ethics

The study was approved by the Research Ethics Committee of the Abadan School of medical sciences (Ethical code:IR.ABADANUMS.REC1398.023), Abadan, Iran. Written informed consent was obtained from all the children’s parents.

3. Results

3.1. Bacterial isolation

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 13(14.3%), 7(7.7%), 57(62.6%) 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 2.

3.2. Antimicrobial susceptibility test

Among 91 Shigella isolates, the highest rates of resistance were to trimethoprim-sulfamethoxazole (87.9%, 80/91), ampicillin(86.8%, 79/91), and tetracycline (80.2%, 73/91). The antimicrobial susceptibility pro file of the Shigella spp. to 10 antibiotics are shown in Table 3. MIC results were as follows: ciprofloxacin (1-256 μg/L),amikacin and gentamicin (0.5-256 µg/L), ceftriaxone (30-256 µg/L),and cefotaxime, ceftazidime (5-256 µg/L). The majority of isolates 76.9% (n=70) were MDR with 20 different patterns.

3.3. Frequency of intI and intII genes

The int

Ⅰ

and int

Ⅱ

genes were detected in 56.0% (n=51) and 86.9% (n=79) strains of Shigella, respectively. None of the isolates had integrin class Ⅲ (int

Ⅲ

) gene. All MDR strains int

Ⅱ

alone or in combination with int

Ⅰ

. The distribution of integrons in different serotype isolates of Shigella is shown in Table 4.

Table 2. Distribution of Shigella spp. by age [n (%)].

Table 3. Frequency of antibiotic resistance among Shigella isolates[(n, %)].

Table 4. Distribution of integrons in different serotype isolates of Shigella [n(%)].

4. Discussion

Shigellosis is a significant public health problem in the world,especially in developing countries and causes 5 to 10% diarrhea in different regions and recently, in Asia, the incidence of this infection cause 414 000 deaths per year[15]. In endemic regions of the developing countries, shigellosis is predominantly a pediatric disease. In our study, the prevalence of shigellosis was 5.9%,which is similar to some studies[16-18], but higher than previous reports[19,20]. It seems that the difference in the distribution of Shigella strains in various studies is due to the difference in geographic and socioeconomic variables, laboratory mistake in identifying isolates, time, and study conditions. The most frequent age group in our study was age 1-5 years, similar to other studies[21,22].The reason might be children in this age group being susceptible to microorganisms, poor hygiene, and lower immune responses in this age group[23]. The geographical distribution of the four Shigella spp.varies in different regions, S. flexneri was the major bacteria that caused diarrhea in most Asian countries[24]. Our study showed that S. flexneri 47 (51.6%) was the predominant species among Shigella strains in Ahvaz and Abadan, which is comparable with previous studies in Iran and other countries[4,17,24], although others studies have shown the most common serotype isolated was S. sonnei[2,4]. Antibiotics are often used for children with bloody and chronic diarrhea to reduce the duration of the disease. Because shigellosis is very contagious, information about the antimicrobial susceptibility is very important for suitable treatment and management of the disease[25].The antibiotic resistance pattern of Shigella spp. varies in different geographic regions. The emergence of MDR strains in Shigella spp.is a growing concern around the world[26]. In this study majority of Shigella isolates were resistant to trimethoprim/sulfamethoxazole(87.9%), ampicillin (86.8%), and tetracycline (80.2%), which is similar to the previous study from Iran and other countries[26-28].According to these results, these antibiotics are not appropriate to treat shigellosis in these regions. The results showed that gentamicin,amikacin, and ciprofloxacin were the best antibiotics against Shigella isolates. The increasing prevalence of MDR to Shigella spp. is a serious problem in developing countries. In our study, the prevalence of MDR in Shigella spp. isolates were (76.9%). Other studies reported a high percentage of MDR to Shigella spp.[16,29],but our results showed that MDR rates were higher than the previous study in the southwest, Iran[17]. It seems that abuse and overuse of antibiotics for the treatment of diarrhea is one of the main causes of high levels of MDR. Antibiotic resistance in Shigella spp. generally occurs due to mobile genetic elements such as transposons, plasmids,and integrons. Mobile genetic elements can cause a distribution of drug resistance genes among different bacteria. MDR in Shigella spp. sometimes can be caused by int

Ⅰ

and int

Ⅱ

genes[14]. In the current study (56%) and (86.9%) of Shigella isolates carried intI and int

Ⅱ

genes, respectively. None of the isolates had the int

Ⅲ

gene. These results are similar to previous studies[24,30,31]. Our results showed that the prevalence of class 2 is significantly higher than in class 1. The results showed that Shigella isolates with both classes of integrons 1 and 2 had a high prevalence of MDR. Also, the prevalence of int

Ⅱ

genes was noticeably associated with MDR in the Shigella isolates. These results suggest that there is a relationship between the int

Ⅱ

gene and other antibiotic-resistant genes that require further studies on molecular level studies. More continuous surveillance studies should be conducted in other parts of the world to investigate the true distribution of Shigella isolates carrying the int

Ⅱ

gene.

In conclusion, antibiotic resistance has increased in Shigella spp.due to misuse and overuse of antibiotics. The high prevalence of multi-drug resistance in Shigella isolates in our area increases the concerns about dissemination of the antibiotic-resistant isolates in this bacterium.

Avoiding the distribution of antibiotic resistance and the spread of the integrons in Shigella spp. is an immediate issue. Therefore,regular monitoring programs to prevent further spread of MDR Shigella isolates is essential.

Conflict of interest statement

The authors report no conflicts of interest in this work.

Acknowledgments

This work was supported by the Vice-Chancellor for Research grant(Grant No. U98-564) 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.

Authors’ contributions

MA developed the original idea and the protocol, performed the experiments, KA was involved in data collection and wrote the preliminary draft, FJ analyzed the data, NJ revised the manuscript,SS was the advisor.