Molecular detection and characterization of Shigella spp. harboring extended-spectrum β-lactamase genes in children with diarrhea in northwest Iran

Shigellosis is one of the acute bowel infections and remains a serious public health problem in resource-poor countries. The present study aimed to survey the distribution of extended-spectrum β-lactamase (ESBL)-producing Shigella strains isolated from patients with diarrhea in northwest Iran. In the present cross-sectional study, from January 2019 to December 2020, 1280 fecal samples were collected from children with diarrhea in Ardabil, Iran. Multiplex PCR assay was applied for the presence of ipaH, invC, wbgZ, rfpB, and rfc genes to detect Shigella spp., Shigella sonnei, Shigella dysenteriae, Shigella flexneri, and Shigella boydii, respectively. Phenotypic detection of ESBL-producing isolates was carried out using the Double Disc Test (DDT). The frequency of main ESBL encoding genes including blaCTX-M, blaSHV, and blaTEM was detected using multiplex PCR. The genetic similarity of S. sonnei isolates was determined using ERIC PCR. A total of 49 Shigella isolates (3.8%; 49/1280) including 42 (85.7%) S. sonnei, 5 (10.2%) S. flexneri, and 2 (4%) S. dysenteriae were identified. S. boydii was not detected in any fecal samples. ESBLs were produced by 10.2% of Shigella spp. including 3 S. sonnei, 1 S. flexneri, and 1 S. dysenteriae. The ESBL encoding genes include blaCTX-M and blaTEM found in 65.3% and 61.2% of isolates, respectively. blaSHV gene was not detected in any isolates. The ERIC-PCR profiles allowed the differentiation of 42 S. sonnei strains into 6 clusters. Our study revealed a high frequency of ESBL-encoding genes among Shigella spp. in northwest Iran. The high prevalence of S. sonnei harboring ESBL genes, in the present work, is the main challenge for dysentery treatment, and this concern justifies the need for effective and regular monitoring of antibiotic usage among patients.


Introduction
Shigellosis is one of the main acute bowel diseases and remains a major public health problem in resourcepoor countries. This disease is caused by gram-negative bacteria belonging to Enterobacteriaceae [1]. In general, Shigella species are facultative intracellular and non-flagellated clinically important pathogens. Shigella flexneri (S. flexneri) with 10 serotypes, Shigella dysenteriae (S. dysenteriae) with 12 serotypes, Shigella boydii (S. boydii) with 18 serotypes, and Shigella sonnei (S. sonnei) with 1 serotype are the four major serological

Open Access
Molecular and Cellular Pediatrics groups of Shigella spp. [2,3]. The infectious dose of Shigella species is very low (10 to 100 organisms), and the fecal-oral route represents the main transmission line to shigellosis [4]. According to several factors such as partially developed immunity, poor hygiene, and lack of past exposures, the age group less than 5 years is highly susceptible to Shigella infections. Among developing countries and in children aged < 5 years, diarrhea is the second most common cause of death [5]. The use of effective antibiotics for the treatment of Shigella infections may lead to a reduction in disease transmission and prevention of lethal outcomes [6]. However, despite the existence of effective treatment regimens, shigellosis continues to be a major global health challenge with estimated shigellosis deaths of 28,000 to 48,000 among young children in 2013 [7]. Antibiotic therapy is usually recommended for dealing with shigellosis because it may shorten the clinical course of the disease, reduce the risk of transmission, and prevent potentially fatal complications [8]. Among the different groups of antibiotics, β-lactams and quinolones are usually used against Shigella infections. However, over the past half-century, many countries have reported the high resistance of Shigella species to common antimicrobial agents [9]. Moreover, based on the geographical location, the antibiotic resistance profile of Shigella spp. varies, and the treatment process is difficult [6,10]. β-Lactams are valuable drugs for treating various types of bacterial infectious diseases [11]. The excessive use of β-lactam antibiotics has led to an increase in resistance to them, especially in gram-negative bacteria. β-lactamases are enzymes that bacteria use to break the β-lactam ring of β-lactam antibiotics and become resistant to them [12,13]. Different types of β-lactamases have been identified, which differ from each other in terms of structural characteristics and molecular targets, and amino acid sequences [14]. Accordance to Ambler's classification, β-lactamases are divided into four groups including A, B, C, and D. Extended-spectrum β-lactamase (ESBL) is a group of β-lactamase that confer resistance to potent β-lactams such as third-generation cephalosporins [15]. ESBL-producing Shigella spp. has been identified as a major concern in hospital and community-acquired infections worldwide, especially in developing county [16]. In this regard, the present study aimed to survey the distribution of ESBL-producing Shigella strains isolated from patients with diarrhea in northwest, Iran.

Study area, sample collection, and bacterial isolates
The present research was performed in Ardabil, an ancient city in northwestern Iran. From January 2019 to December 2020, 1280 fecal samples were collected from children with diarrhea who had been referred to the laboratory of Bu Ali Hospital at Ardabil University, belonging to the Iranian health system in Ardabil, Iran. The fecal samples were cultured on MacConkey agar and selenite F medium, and all plates were incubated overnight at 37 °C. After incubation time, all colonies were transferred to Hektoen enteric agar (HE) and xylose-lysine deoxycholate agar (XLD agar), (Merck, Hamburg, Germany) and were incubated at 37 °C for 18 to 24 h. Specimens with green colonies on HE medium, colorless colonies on XLD medium, and non-lactose fermenting colonies on Mac-Conkey agar were suspected of Shigella species. The final identification of Shigella species was conducted using conventional biochemical tests such as triple sugar iron (TSI), SIM medium (sulfide indole motility medium), ornithine decarboxylase (ODC), lysine iron agar (LIA), Simmons citrate, and urea agar. In the next step, all confirmed bacteria were stored at −80 °C in 10% glycerol ( Fig. 1).

Molecular detection of Shigella species
DNA was extracted using the DNA extraction kit (Sinaclon, Tehran, Iran) according to the manufacturer's instructions. The NanoDrop device (Thermo Fisher Scientific, Waltham, MA, USA) was used for the evaluation of the quality and quantity of the extracted DNA. Molecular identification of Shigella species was performed using multiplex PCR assay. The specific genes including ipaH, invC, wbgZ, rfpB, and rfc were employed to detect Shigella spp., S. sonnei, S. dysenteriae, S. flexneri, and S. boydii, respectively [17,18]. The sequence results were submitted in GenBank (accession numbers: MN503255.1 and MN503256.1).
The sequence of the primers used for multiplex PCR assay is shown in Table 1. The PCR reaction was performed in a thermocycler (Bio-Rad, Germany) as follows: 1 cycle at 94 °C for 4 min, 32 cycles at 94 °C for 45 Moreover, DNase-free as a negative control was used for PCR assay. The PCR products were stained by safe stain and screened by electrophoresis on 1-1.5% agarose. The bands associated with PCR products were observed through the application of a transilluminator.

Phenotypic detection of ESBL-producing isolates
ESBL production was determined according to the Clinical Laboratory Standards Institute (CLSI) guidelines [21]. Phenotypic detection of ESBL-producing isolates was carried out using ceftazidime (CAZ) and cefotaxime (CTX) disks (Becton Dickinson) and by the double-disk test on freshly prepared Mueller-Hinton agar (Fig. 2). Briefly, CAZ and CTX 30 μg disks, with and without clavulanic acid (CA) 10 μg disk, were used for testing. An inhibition zone ≥ 5 mm for CAZ or CTX tested in combination with CA versus its zone when tested alone was considered as ESBL-producing isolates. Escherichia coli ATCC 25922 was used as the standard strain.

Molecular detection of ESBL encoding genes
A multiplex PCR assay targeting the main ESBL encoding genes including bla TEM , bla CTX-M , and bla SHV was performed. The sequence of primers used for multiplex PCR assay is shown in

Statistical analysis
The data were inputted and analyzed using the SPSS software ver. 23 (SPSS Inc., Chicago, IL, USA). A binary method and ward method were used for computing distance matrix and hierarchical clustering, respectively. The cophenetic value was determined as 0.647; this index measures the correlation of cophenetic (height) distance to the original distance in the data. To assess clustering tendency (the feasibility of the clustering analysis), Hopkins statistics was used, and its value was less than 0.5 (Hopkins statistic = 0.4), indicating that we could perform cluster analysis on the data set. All statistical analyses were performed in R software.

Results
In the present study, a total of 1280 fecal samples were screened for Shigella genus. Conventional biochemical tests were employed for the identification and differentiation of Shigella spp. Based on biochemical tests, Shigella spp. was detected in 8.8% (n = 113/1280) of the fecal samples (Fig. 1). The prevalence of Shigella species among children with diarrhea is summarized in Table 2.
Overall, Shigella species were isolated from 3.8% (n = 49/1280) of the samples using PCR assay (Fig. 3). The prevalence of Shigella species is given as follows: S. sonnei  Table 2.
The ESBL encoding genes include bla CTX-M and bla TEM found in 65.3% and 61.2% of isolates, respectively (Figs. 4 and 5). bla SHV gene was not detected in any isolates. The frequency of ESBL encoding genes among Shigella species is shown in Table 2.

ERIC PCR analysis
The genotyping profiles of 42 isolates of S. sonnei strains according to ERIC-PCR fingerprinting are shown in Figs. 6 and 7. All 42 S. sonnei under analysis produced 6-10 amplicons ranging from 100 to 1500 bp. The ERIC-PCR profiles allowed the differentiation of 42 S. sonnei strains into 6 clusters.

Discussion
Among children in developing countries, despite the existence of effective treatment regimens, shigellosis continues to be the main public-health concern with an annual estimate of 163.2 million new cases and 1.1 million deaths [22]. In Iran, there are no specific guidelines to employ antibiotic therapy, and in most cases, physicians prescribe antibiotics without any stool cultures [23]. Therefore, performing epidemiological studies and gathering information regarding phenotypic and molecular mechanisms of antibiotic resistance to control infections and the development of local treatment guidelines are necessary.
In the present study, we surveyed the prevalence and molecular characterization of Shigella species harboring ESBL genes in patients with diarrhea from the northwest of Iran. This study showed a prevalence of 3.8% (49/1280) of shigellosis that was lower than the rates found in previously published studies conducted by Farajzadeh Sheikh et al. [6], Soltan Dallal et al. [24], Abbasi et al. [25], and Ranjbar et al. [20]. All of these studies were performed in Iran, and it was reported that the prevalence of Shigella  [28]. These studies found that the frequency of Shigella species in the patients with diarrhea was 2.1%, 1.9%, 5.9%, and 1.3%, respectively.
The detected variation in the prevalence of Shigella spp. could be due to the difference in the sample collection seasons, geography, specimen size, diversity of specimen type, study population, and applied detecting methods [27]. The result of the present study indicates that shigellosis is the main public health problem in the northwest of Iran. Therefore, it is critical to take several measures such as promoting awareness about the safety of water and food and improving health conditions.
Our finding revealed that S. sonnei had the highest frequency among Shigella species. In contrast, S. boydii was not detected in fecal samples. This result was in line with previous reports from Farajzadeh Sheikh et al.    [31]. According to previous studies, it seems that in the southern regions of Iran, in the cities such as Shiraz, Ahvaz, and Kerman, S. flexneri is the dominant strain, while in northern regions of Iran, S. sonnei strain is prevalent [27,[32][33][34][35][36]. In contrast with our results, several different studies in various countries including Nepal [24], Ethiopia [37], Bulgaria [38], India [22], Iran [34], and Peru [39] have reported the S. flexneri as the leading cause of shigellosis.
In general, Shigella species can cause several diseases such as diarrheal in both developed and developing countries [40]. It has been reported that S. flexneri is the predominant serogroup in developing countries and is responsible for 44.5-80% of all Shigella infections such as diarrhea, while S. sonnei is a major cause of diarrheal in industrialized and developed countries [30].
In the present study, 10.2% were ESBL-producing strains by the double-disk method. Most ESBL-producing species were detected in S. sonnei which was following studies reported by Ranjbar [41]. In contrast, in a study performed by Aminshahidi et al., 54.5% of S. flexneri strain was ESBL positive [33]. Several different studies have surveyed the frequency of ESBL-producing Shigella strains. Results of a study performed by Li et al. from China revealed that ESBLs were produced by 18.1% of Shigella isolates, and most of the ESBL-producing species belonged to the S. flexneri isolates (19.5 %) [15]. In another study, Zhang et al. in China revealed that 10 Shigella isolates produced ESBLs including 8 S. sonnei isolates and 2 S. flexneri isolates [30].
The detected variation in the frequency of ESBL-producing Shigella species could be due to the differences in the applied detecting methods, variations in geographical location, and differences in sample size, sample type, and study participants [44]. Mobile genetic elements such as integrons, plasmids, and transposons can transmit the drug resistance genes among different bacteria and are responsible for antibiotic resistance in Shigella spp. [25]. In general, the main aim of antibiotic prescription in children with bloody and chronic diarrhea is the reduction in the duration of the disease. Because most Shigella infections are contagious and severe, appropriate antibiotic prescription and suitable treatment are essential [27].
Recently, the emergence of multidrug-resistant (MDR) Shigella species is increasing. The treatment of MDR strains is very difficult and is considered an alarming public health concern, worldwide [29].
In the present study, the bla CTX-M with a frequency of 65.3% was the most predominant ESBL encoding gene followed by the bla TEM [20].
β-Lactamases are the enzymes encoded by several genes and were considered the main mechanisms of resistance to β-lactam antibiotics such as cephalosporins (especially third-generation cephalosporins) among gram-negative bacteria. The high prevalence of ESBL-producing and β-lactamase genes leads to complicated treatment [33,45].
Identification of ESBL among Shigella strains is an undeniable concern because it reduces antibiotic treatment options, and the spread of mobile resistance determinants will be a great threat to the treatment of invasive diseases in the future.
The present study has several limitations including the following: [1] the antibiotic susceptibility of Shigella species against different classes of antibiotics such as carbapenems was not determined, [2] the frequency of virulence genes was not determined, [3] the prevalence of other antibiotic resistance encoding genes such as genes encoding AmpC β-lactamase was not examined, and [4] the patients' demographic data such as age, underlying disease, duration of hospitalization, and the outcome of treatment are not accessible; therefore, we were unable to perform deep analyses.

Conclusion
The present study elucidated an update on the phenotypic and genotypic prevalence of ESBLs appearing among Shigella species circulating in the northwest of Iran. The high prevalence of Shigella species, especially S. sonnei, harbored ESBL genes in the present work; this is the main challenge for dysentery treatment, and it highlights the need for effective and regular monitoring of antibiotic usage among patients. Therefore, continued surveillance of the antimicrobial resistance profile and monitoring of the prevalence of Shigella speciesproducing ESBLs in Iran are urgently required. Epidemiological studies such as the present study provide valuable data on indigenous and resistant strains which help identify sources of infection, improve infection control systems, administrate effective drug treatment, and increase public health in the human community.