https://orcid.org/0000-0002-9828-8609
Abstract
Background
Shigella spp. are primary pathogens of diarrhea in children worldwide. Emergence of resistance to fluoroquinolones and third-generation cephalosporins is crucial in the management of pediatric shigellosis. We determined the prevalence and the antibiotic resistance patterns of Shigella species isolated from pediatric patients in central Iran.
Materials and methods
Pediatric diarrhea samples (n=230) were cultured on MacConkey and XLD agar media and in GN broth. Genus-specific PCR for ipaH was also used for detection directly from fecal specimens. Antibiotic resistance and the frequency of ESBL and AmpC genes were determined.
Results
Out of the 230 samples, 19 (8.2%) cases of Shigella spp. were identified using culture. Twenty-six samples were positive by PCR (11.3%), S. flexneri (4/19; 21%) and S. sonnei (15/19; 78.9%) being the most detected. The highest antibiotic resistance rates were found for cotrimoxazole (19/19; 100%), ampicillin (16/19; 84.2%), cefixime (13/19; 68.4%) and ceftriaxone (12/19; 63.1%). Ten cases showed phenotypic ESBL presence and all these strains were positive for blaTEM, blaCTX-M-1, and blaCTX-M-15. Three strains were AmpC positive, all of which harbored blaCMY-2 and two contained blaCIT. Of the 19 Shigella isolates 5 (26.3%), 2 (10.5%), and 1 (5.2%) were phenotypically resistant to nalidixic acid, ciprofloxacin, and norfloxacin, respectively. Class 1 integron was found in 18 (94.7%) isolates whereas class 2 integron was found in 19 (100%) strains.
Conclusion
We found a considerable presence of Shigella species with elevated antibiotic resistance levels. In particular, the resistance to third-generation cephalosporins (ESBL) and ciprofloxacin must be taken seriously.
Introduction
Shigella species belong to the Enterobacteriaceae and are Gram-negative rods, known as important causative agents of diarrhea and dysentery. Shigella spp. pose major public health problems in developing countries.Citation1 In 2013, the annual and global number of deaths amongst children under 5 years due to shigellosis was estimated to be between 28,000 and 48,000.Citation2 The most prevalent species causing those infections are Shigella flexneri and Shigella sonnei.Citation3 Due to resistance to trimethoprim-sulfamethoxazole, ampicillin, sulfonamides, and tetracycline World Health Organization (WHO) recommends fluoroquinolone (ciprofloxacin) for first-line treatment of all patients with bloody diarrhea, regardless of age. Ceftriaxone is proposed as second-line therapy or alternative antimicrobial agent in adults and children.Citation4 The increase of multidrug resistance (MDR) in Shigella spp., mostly due to third-generation cephalosporins (TGC), azithromycin and fluoroquinolones, is a major issue in developing countries.Citation5 The elevated resistance levels are often the consequence of the horizontal transfer of complex resistance determinants including plasmids, integrons, and transposons.Citation6
Although studies have found different frequencies and different Shigella species from north and south Iran,Citation7–Citation9 most reported phenotypic criteria such as frequency and antibiotic resistance profiles. In addition, Iran is a huge country with different climates in different geographical regions. In Central Iran, the prevalence of diarrhea and dysentery among children is especially high in the summer. Still, laboratory diagnosis of Shigella spp. is not common and most patients are treated empirically with TGCs.
Therefore, the main objective of the present study was to determine the frequency of Shigella spp. in children with diarrhea in Central Iran. In addition, we investigated their phenotypic antimicrobial resistance levels and resistance gene content.
Materials and methods
Ethics statement
This study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of the Arak University of Medical Sciences under approval numbers ARAKMU.REC. 93–176-30. A questionnaire and a consent form were provided to the parents, guardians and/or patients. The two main inclusion criteria were as follows: 1) completion of the consent form; and 2) direct observation of over five white blood cells per high-power microscope field (HPF) in a diarrhea sample.Citation7 None of the patients had taken antibiotics in the week before hospitalization.
Sample collection
This descriptive cross-sectional study included samples from 230 pediatric patients with infectious diarrhea who were referred to the Educational-Therapeutic Centers affiliated with the Arak University of Medical Sciences (Valiasr, Amirkabir, Amiralmomenin) from May to September 2015.
Phenotypic investigation
First, 500 μL of the liquid stool samples were enriched in 5 mL of Gram-negative (GN) broth (Merck, Hamburg, Germany) and after 6 h at 37°C the enriched samples were streaked on xylose lysine deoxycholate (XLD) and MacConkey agar. In addition, stool samples were directly cultured on MacConkey agar, XLD (Merck) and incubated at 35–37°C for 18–24 h. Individual isolates were surveyed using previously defined standard biochemical and serological tests. All isolates were confirmed to be Shigella spp. applying API 20E test strips (bioMerieux, Craponne, France). Serogrouping of the Shigella spp. isolates was performed using slide agglutination with specific antisera (SSI, Copenhagen, Denmark).Citation8 The confirmed Shigella spp. isolates were kept in brain heart infusion broth with 20% glycerol at −80°C. S. sonnei PTCC 1777 and S. flexneri PTCC 1234 were used as controls in each assay (obtained from the Iranian Research Organization for Science and Technology). S. boydi and S. dysenteriae control strains were obtained from the microbiology department of Arak University of Medical Sciences.
Investigating Shigella antibiotic resistance by disk diffusion
Antibiotic resistance of Shigella spp. isolates was determined using the disk diffusion method according to the Clinical and Laboratory Standards Institute (CLSI) 2016 guidelines.Citation9 The antibiotic discs used contained cotrimoxazole (25 μg), cefoxitin (30 μg), ampicillin (10μg), ceftizoxime (30 μg), ceftazidime (30 μg), ceftriaxone (30 μg), cefixime (5 μg), cefotaxime (30μg), tetracycline (30 μg), gentamicin (10 μg), nalidixic acid (30 μg), chloramphenicol (30 μg), ciprofloxacin (5 μg), azithromycin (15 μg) and imipenem (10 μg) (Mast Diagnostics, Bootle, UK).
Detection of extended spectrum β–lactamase (ESBL) and AmpC by phenotypic methods
Shigella spp. isolates were subjected to double-disk synergy testing, combination disk diffusion methods, and AmpC disk testing and phenyl boronic acid assays for identification of ESBL and AmpC resistance according to the 2016 CLSI guidelines.Citation9 The MICs of 19 of the isolates to ciprofloxacin were determined by the E-test (Liofilchem, Roseto degli Abruzzi, Italy).
Genotypic investigations
DNA extraction
DNA extraction was performed directly from the stool samples and the reference S. sonnei and S. flexneri isolates using the QIAamp DNA Stool Minikit (Qiagen GmbH, Hilden, Germany) according to the manufacturer’s protocol. The amount and purity of extracted DNA were measured using a NanoDrop device (Thermo Fisher Scientific, Waltham, MA, USA).
Genotypic identification
The ipaH gene was used as a genetic marker for confirming the Shigella genus by PCR.Citation10 PCR assays were performed to detect the stx gene for Shiga toxin-producing isolates and the ESBL genes (blaTEM, blaSHV, and blaCTX-M-1, 2, 8, 14, 15) for the ESBL-producing Shigella spp. isolates. In addition, AmpC genes (blaCMY-2, blaCIT, blaACC, blaFOX, blaMOX, and blaDHA) were also detected by PCR.Citation11–Citation13 PCR was carried out to amplify plasmid-mediated quinolones resistance (PMQR) targets for qnr determinants including qnrA, qnrB, and qnrS. Mutations in gyrA and parC among the fluoroquinolone-resistant Shigella spp. isolates were identified with the use of DNA sequencing as well.Citation11,Citation13
Integron detection
To detect and distinguish class 1, 2 and 3 integrons, PCR analysis was performed as described before.Citation3,Citation10,Citation11
Results
Out of the 230 pediatric infectious diarrhea samples, 19 (8.2%) cases of shigellosis were identified using the culture method. All culture-positive samples were identified as positive using the PCR method and an additional seven samples which were culture-negative were identified as positive using the PCR method with specific primers 26 (11.3%). Of 230 patients included in the study, 10 (52.6%) male and 9 (47.3%) female patients were infected by Shigella spp., giving an infection ratio of males to females of 1.1:1. The average age of the patients was 6 years and 4 months. The youngest of these patients was a boy aged one year and 10 months, and the oldest was a 16-year-old boy. The majority of Shigella spp. infections (7 cases, 36.8%) were observed in pediatric patients between 5 and 10 years of age. Clinical symptoms of pediatric patients are given in .
Table 1 Classification of age groups and clinical symptoms of pediatric patients infected by Shigellaspp
Phenotypic and genotypic investigation
Of the 19 cultured Shigella spp. isolates, 15 (78.9%) were identified as S. sonnei and 4 (21%) as S. flexneri. No Shiga toxin-producing isolate was found.Citation14
Phenotypic and genotypic antibiotic resistance determination
Using the CLSI 2016 guidelines, the highest resistance rates in Shigella spp. were found against cotrimoxazole (19/19; 100%), ampicillin (16/19; 84.2%), cefixime (13/19; 68.4%), ceftriaxone (12/19; 63.1%), and cefotaxime (12/19; 63.1%). All Shigella isolates were susceptible to azithromycin and imipenem (). Ten Shigella spp. isolates were ESBL positive. All isolates contained blaTEM, blaCTX-M-1, and blaCTX-M-15 resistance genes. In addition, all AmpC positive isolates (3/19; 15.8%) were blaCMY-2 positive and were resistant to ciprofloxacin and nalidixic acid; only 2 (66.6%) were blaCIT type positive. (). Of the 19 Shigella isolates studied, 5 (26.3%), 2 (10.5%), and 1 (5.2%) showed resistance to nalidixic acid, ciprofloxacin, and norfloxacin, respectively. Among the PMQR determinants, qnrS was positive only in 60% (3/5) of the isolates. In addition, no isolates harbored qnrA or qnrB genes. The majority of the Shigella spp. isolates (89.4%) were multidrug resistant (). All the isolates harboring PMQR had the same mutations in gyrA at amino acid 83 (replacement of serine with leucine) and in parC at amino acid 80 (replacement of serine with isoleucine; GenBank accession no. HM068910). Out of 5 (26.3%) fluoroquinolone-resistant isolates, 4 (80%) were S. sonnei and 1 (20%) was identified as S. flexneri. Also, 3 of the fluoroquinolone-resistant isolates (two S. sonnei and one S. flexneri) were resistant to the TGC and fourth-GC and presented AmpC producer. Class 1 integrons were found in a total of 18 (94.7%) strains: S. sonnei (n=14) and S. flexneri (n=4), whereas class 2 integrons were found in all strains: S. sonnei (n=15) and S. flexneri (n=4); Class 3 integrons were not found ().
Table 2 Frequency of antibiotic resistance among Shigella isolates
Table 3 Frequency of antibiotic resistance genes among Shigella isolates
Table 4 Phenotypic and genotypic antibiotic resistance rates in Shigella spp
Discussion
In total, an 8.2% prevalence of Shigellosis was defined using cultures and 11.3% using PCR. The sensitivity of the PCR method is higher than those of the culture methods.Citation15,Citation16 However, our result was almost similar to studies conducted in Kerman, Iran and the North of Ethiopia, which had reported frequencies of 9% and 13.3%, respectively.Citation17,Citation18 Our result was different from studies conducted in India which had reported frequencies close to 2%.Citation19 These differences in frequency of Shigellosis may be related to age, economic development level, geography, climate as well as many other environmental conditions. Facilities such as general water supply systems and sewage systems, closely related to the level of sanitation and individual hygiene, contamination of food, and improper medical health-care levels may be missing or of poor quality.
S. sonnei is more generally found in industrialized countries, whereas S. flexneri seems overrepresented in the developing world.Citation1 Our results show that S. sonnei was the major species in this study and comprised 78.9% of all Shigella spp., followed by S. flexneri with 21%. This finding is similar to recently reported data from Iran (Shiraz)Citation20 as well as other countries including Thailand.Citation20 However, this pattern is different from other studies in Iran (Abadan), south of IranCitation21 and countries such as IndiaCitation22 where the most common species was S. flexneri. During recent decades, most cases of shigellosis in Iran were caused by S. flexneri, although recent studies have shown an increasing number of infections caused by S. sonnei.Citation20,Citation23 Socioeconomic conditions and general hygiene affect the frequency of Shigella spp. serogroups.Citation24
In this study, no shiga toxin-producing isolate was found. In similar studies, prevalence of stx gene was reported to be 1.4% in S. dysenteriae in Tehran (Iran), 3 (1.4%) of S. flexneri isolates in Canada and 21% S. flexneri and S. dysenteriae in France.Citation25–Citation27
Because shigellosis is highly contagious, information about the frequency of the disease and the antimicrobial susceptibility of the strains is important to ensuring suitable clinical treatment and patient management.Citation28 Mild symptoms are self-limiting, but antibiotics are advised to reduce the duration of diarrhea in case of severe dysentery cases. The antibiotic resistance properties of Shigella spp are various, and regionally distinct.Citation29 We provide the first report regarding the prevalence of shigellosis and associated resistance patterns to a panel of 16 antibiotics in the center of Iran. The appearance of multidrug-resistant (MDR) strains of Shigella spp is a growing concern across the globe.Citation11
Cotrimoxazole is a drug often used for empirical therapy of diarrheal diseases; extensive use of this drug has led to the advent of resistant Shigella spp. strains.Citation30 In this study, Shigella spp. showed a high rate of resistance to cotrimoxazole (100%). Previous reports in Iran have reported a 92.2% to 94% resistance level to cotrimoxazole (2000–2017).Citation30,Citation31 A high-level resistance to cotrimoxazole has also been reported from Turkey (95%).Citation32
Based on the reports from our region, resistance to ampicillin ranged from 12–20% to 87%.Citation33,Citation34 We here document 84.2%, and a previous study reported 57% from Iran.Citation35 These results strongly suggest that cotrimoxazole and ampicillin can no longer be empirically used for the treatment of severe diarrhea and dysentery in central Iran.
A previous Iranian study from Tabriz found that 4.2% of Shigella spp. isolates were resistant to ciprofloxacin.Citation11 Ciprofloxacin-resistant S. flexneri was also recognized from parts of India (56.2%).Citation19 In the present study, 5 (26.3%) of Shigella isolates were resistant to nalidixic acid, a marked increase in resistance to nalidixic acid. In two reports from Tehran and Tabriz, Iran, 17.4% and 31% of Shigella spp. isolates were resistant to nalidixic acid, respectively.Citation11,Citation36 In China, Pu et alCitation37 found that 5 (33.3%) of 15 fluoroquinolone-resistant isolates contained the qnrS gene. The findings of the current study regarding other PMQR genes (qnrA and qnrB) are in accordance with the findings of Taneja et al,Citation38 who explained that none of the Shigella spp. isolates they found were positive for qnrA and qnrB genes.
In this study, high resistance (21–68.4%) to TGC was observed in the Shigella spp. isolates. Previous studies from Iran have also indicated the resistance of Shigella spp. to cephalosporins at the range of 7.3–57.7% between 2008 and 2018.Citation11 This indicated that the rate of resistance to cephalosporins is growing among Shigella spp. in Iran, similar to what is happening in other countries.Citation39 This finding is alarming and contrary to previous studies from China, the Middle East, and Southeast Asia which have reported lower level resistance to cephalosporins (2.0–15.1%).Citation11,Citation12
In the present investigation, blaTEM, blaCTX-M-1, and blaCTX-M-15 were the most common ESBL genes, and blaCMY-2 and blaCIT were the most common AmpC genes. The current report shows that resistance to β-lactams mediated by blaCMY enzymes in Iran has a similar pattern as in other parts of the world. In recent years, various ESBL-producing Shigella were also reported from Argentina (blaCTX-M-2), Korea and China (blaCTX-M-14), and Vietnam (blaCTX-M-15 and blaCTX-M-24).Citation40–Citation42 blaCMY-2 type AmpC ß-lactamase producers were predominant. However, in several other studies, the blaCMY-2-type AmpC ß-lactamases were the most prevalent cephalosporinases in Iran (Tehran) and Taiwan.Citation43,Citation44
In this study, class 1 integrons were found in 18 (94.7%) strains, whereas class 2 integrons were found in 19 (100%) of the strains. In Brazil, it was found that class 1 integrons were achieved in only two strains, whereas class 2 integrons were achieved in 56 (90.3%) of the strains.Citation45 Of the three classes of integrons linked to antimicrobial resistance, the class I integron is the most frequently found in Gram-negative bacteria.Citation46 The class II integron is the most predominant integron in S. sonnei.Citation47 The prevalence of integrons in the Enterobacteriaceae family has been varied and played an important role in the development of drug-resistant bacteria. Therefore, a high prevalence of antibiotic resistance is probably related to a high prevalence of class I and II integrons. In epidemiologic studies, particularly of infectious diseases caused by bacteria, typing of the isolates is a useful method for source tracing and understanding the pathogenesis and eventual disease prevention. One limitation of the present study was that typing of Shigella isolates was not performed.
Conclusion
The recent emergence of S. sonnei in developing countries reinforces the need for effective epidemiological surveillance systems.Citation48 The wide distribution of MDR Shigella spp. isolates and the continuing emergence of ESBL-producing and ciprofloxacin-resistant isolates were shown in the study. The high prevalence of ESBL-producing genes in Shigella spp. isolates in pediatric patients in our study can be a major challenge for dysentery treatment. We found that the frequency of ESBL producing Shigella spp. isolates was higher than those in many other countries. Our results enhance concerns about the dissemination of ESBL among the strains of endemic S. sonni throughout the country, because it is now the most frequently isolated Shigella species in Iran. Thus, to prevent outbreaks due to these resistant isolates, the surveillance of the antimicrobial resistance of Shigella spp. isolates should be continuously considered, and empiric antibiotic therapy should be adapted appropriately.
Abbreviation list
ESBL, third generation cephalosporins; MDR, multidrug resistance; TGC, third generation cephalosporins; GN, Gram-negative; XLD, xylose lysine deoxycholate; CLSI, Clinical and Laboratory Standards Institute; PMQR, plasmid mediated quinolones resistance.
Acknowledgments
This work was conducted with financial assistance from Arak University of Medical Sciences, Iran, and we would like to appreciate their contribution. This paper is extracted from a dissertation by Elnaz Abbasi to fulfill the requirement for Master of Sciences in Medical Bacteriology at Arak University of Medical Sciences, Iran.
Disclosure
AvB is an employee of bioMerieux, a company designing, developing and selling infectious disease tests. The company had no influence on the design and execution, either of the study or in the writing of the manuscript. The authors report no other conflicts of interest in this work.
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