Determination of ESBLs, pAmpC-beta-lactamase genes, and plasmid replicon types among Shigella species from different cities in Iran

1. Introduction

Foodborne bacterial pathogens such as Shigella species (spp.) are among the most critical public health concerns around the world ( 1 ). The Shigella spp., including Shigella dysenteriae, Shigella flexneri, Shigella sonnei, and Shigella boydii are members of the Enterobacteriaceae family and cause acute gastroenteritis with high morbidity and mortality, especially in children in developing countries ( 2 ). Shigellosis, caused by Shigella spp., is an acute enteric infection that is usually characterized by watery and bloody mucoid diarrhea. It is highly contagious due to its low infectious dose. Antibiotic therapy, usually used for infants, the elderly, and immunocompromised patients, can reduce the duration and severity of shigellosis, as well as and the risk of transmission.

Ciprofloxacin, pivmecillinam, ceftriaxone, and azithromycin are antibiotic agents used for the treatment of shigellosis, especially in patients with bloody diarrhea ( 3 ). However, antibiotic resistance is increasing among Shigella spp. due to the misuse or overuse of antibiotic agents in the treatment of shigellosis, and multidrug resistant (MDR) Shigella isolates have been reported in some countries ( 4 ). The ability of this organism to acquire different antibiotic resistance genes through mobile genetic elements such as integrons, transposons, and plasmids is a major factor in the spread and emergence of MDR isolates.

Most MDR Shigella isolates are resistant to cephalosporins, ciprofloxacin, and azithromycin through acquired plasmid-mediated resistance mechanisms ( 4 ). Detection of ESBL- and AmpC- positive Shigella spp. is important because they are usually MDR and therapeutic options for them are limited. Hence, updating our data on the rate and mechanisms of resistance to antibiotic agents in Shigella spp. is essential for using effective and justified therapy to decrease the morbidity and mortality rates associated with shigellosis. Conjugative plasmids play an important role in the evolution and dissemination of antibiotic resistance and virulence factors among bacteria through facilitation of horizontal gene transfer ( 3 ). Due to the importance of Shigella spp. in causing gastrointestinal infections and the significant challenges in the treatment of infections resulting from MDR isolates, this study was conducted to investigate the presence of ESBLs, pAmpC beta-lactamase genes, and plasmid replicon types among Shigella isolates in different cities in Iran.

2. Materials and Methods

2.1. Sampling, Culture, and Shigella spp. isolates

This study was performed on 210 clinical isolates of Shigella spp. that were collected from patients with diarrhea (A single specimen each) and kept at -80 °C in Tryptic Soy Broth (TSB) supplemented with 30% glycerol from 2019 to 2021. The bacterial isolates were obtained from six cities in Iran--Ahvaz, Kerman, Shahr-e Kord, Tabriz, Urmia, and Ardabil-in Iran, and were confirmed using standard microbiological tests and polymerase chain reaction (PCR) with specific primers. The sequences and annealing temperatures of the primers are shown in Table 1.

2.2. Antimicrobial susceptibility of isolates and detection of ESBL-producing isolates

Disk diffusion method was used to evaluate the antibiotic susceptibility pattern of the isolates to different antibiotic agents (Padtan Teb Laboratory Instruments Co., Iran), including ceftazidime (CAZ, 30 μg), ampicillin (AMP, 10 μg), cefotaxime (CTX, 30 μg), cefoxitin (FOX, 30 µg), ceftriaxone (CRO, 30 μg), gentamicin (GEN, 10 µg), levofloxacin (LEV, 5 μg), amikacin (AMK, 30 μg), streptomycin (STR, 10 µg), ciprofloxacin (CIP, 5 μg), chloramphenicol (CHL, 30 μg), nalidixic acid (NAL, 30 µg), azithromycin (AZM, 15 μg), tetracycline (TET, 30µg), and trimethoprim-sulfamethoxazole (SXT, 1.25/23.75 μg), according to the Clinical and Laboratory Standards Institute (CLSI) guidelines ( 5 ). Shigella isolates that were resistant to one of the third-generation cephalosporins-including ceftazidime, cefotaxime, and ceftriaxone-were selected for confirmation of ESBL production according to CLSI guidelines, using the combination disc test with clavulanic acid ( 5 ).

2.3. DNA extraction

The DNA of the isolates was extracted using the boiling method. Briefly, a single colony from each isolate was suspended in 400 µL of DNase- and RNase-free water and heated at 100°C for 10 minutes. The lysates were then centrifuged at 12,000 × g for 10 minutes and the supernatants were used as the DNA template for PCR and multiplex PCR assays.

2.4. Detection of ESBLs and pAmpC among Shigella spp

ESBL resistance genes, including bla_TEM, bla_SHV, and bla_CTX-M, were screened using PCR among ESBL-positive isolates. Cefoxitin-resistant isolates were also selected for identification of plasmid-mediated AmpC beta-lactamase genes, including bla_MOX, bla_ACC, bla_FOX, bla_CMY, bla_EBC, and bla_DHA, using a multiplex-PCR amplification technique previously described by Pérez-Pérez and Hanson ( 6 ). The PCR products were electrophoresed on 1.5% agarose gel for 45 minutes at 100 V, and the gels were analyzed usung a gel documentation imaging system. The sequence and annealing temperatures of the primers used for the detection of beta-lactamase genes are presented in Table 1.

2.5. Plasmid replicon type profiling

PCR-based plasmid replicon typing (PBRT) was performed based on the Carattoli et al., method, using 18 pairs of specific primers to identify replicons FIA, FIB, FIC, HI1, HI2, I1-Iγ, N, L/M, P, W, A/C, T, K, B/O, X, Y, FIIAs, and Frep in five multiplex-PCR and three simplex-PCR ( 7 ). The sequence of primers used in PBRT techniques is presented in Table 2.

2.6. Statistical analysis

The results were statistically evaluated using SPSS software (version 24) and P-values ≤ 0.05 were considered statistically significant, based on the chi-square test and Fisher’s exact test.

3. Results

3.1. Shigella spp. isolates

In the present study, a total of 210 Shigella spp. isolates were collected from 6 different cities, including the cities Kerman [n= 64 (30.4%)], Ahvaz n=67 (33.5%)], Ardabil [n=18 (8.5%)], Urmia [n=19 (9%)], Tabriz [n=20 (9.5%)], and Shahr-e Kord [n=22 (10.4%)], where S. dysenteriae [n=5 (2%)], S. flexneri [n=102 (49%)], S. boydii [n=28 (13%)], and S. sonnei [n=75 (36%)] were reported. The prevalence of Shigella spp. in different cities in Iran is shown in Table 3.

3.2. Result of antimicrobial susceptibility

In this study, the resistance rate to antibiotic agents was as follows: trimethoprim-sulfamethoxazole (97%), streptomycin (94%), ampicillin (80%), tetracycline (77%), chloramphenicol (62%) and cefotaxime (57%), ceftriaxone (49%), nalidixic acid (31%), ceftazidime (30%), gentamicin (13%), azithromycin (12%), levofloxacin (6%), amikacin (6%), ciprofloxacin (6%), and cefoxitin (5%). Interestingly, all isolates were resistant to at least three classes of antibiotics and were considered MDR isolates. The rate of antibiotic resistance in different cities is presented in Table 4.

3.2. Prevalence of ESBLs and pAmpC beta-lactamase genes (bla genes)

Based on the results, 114 isolates (54%) of the isolates were ESBL-positive, and bla_TEM and bla_CTX-M were detected in 84 (40%), and 93 (44%) of ESBL-positive isolates, respectively. bla_DHA as one of the pAmpC-associated genes, was detected only in three isolates (1.4%): two isolates belonged to S. flexneri and one to S. sonnei. The prevalence of β-lactamase genes among Shigella spp. in different cities in Iran is shown in Table 5.

3.3. Prevalence of plasmid replicon types

In this study, the replicon types I1-Iγ (72.8%), K (54.2%), W (47.1%), FIB (30.9%), Y (21.9%), P (13.3%), FIC (6.6%), FIA (4.2%), HI1 (1.9%), and B/O (1.4%) were reported. The Distribution of bla genes and plasmid replicon types among Shigella spp. in Kerman, Ardabil, Shahr-e Kord, Tabriz, Ahvaz, and Urmia, Iran, is presented in Tables 6 and 7.

4. Discussion

Shigellosis poses a considerable threat to global public health, with a particularly high prevalence in developing countries. Administering antibiotics has been shown to reduce both the severity and duration of the infection, as well as the excretion of the organism in feces, thereby aiding in the prevention of its continued transmission. This study offers insights into the molecular epidemiology of antibiotic resistance profiles, ESBLs, AmpC, and plasmid replicon types among Shigella spp. isolated from diarrheal samples in Iran. Top of Form.

In the current study, S. flexneri (49%) and S. sonnei (36%) were identified as the most common species, and our results were largely consistent with the results of recent studies in Iran and various countries ( 8 - 10 ). Between 2001-2019, several studies identified S. flexneri and S. sonnei as the predominant species of Shigella in Ahvaz and Tehran, Iran ( 8 , 9 ). Given that S. flexneri and S. sonnei are typically predominant in developing and developed countries, respectively, the findings of aforementioned studies are consistent with those of our own. The results of the current study showed that despite the high standard of hygiene in our region in recent years, we observed no noticeable change in the prevalence and population composition of isolated Shigella spp.

In recent years, an epidemiological transition in the prevalence of Shigella serogroups has been observed. Specifically, there has been a notable emergence of S. sonnei in regions where S. flexneri previously prevailed. This shift has been documented across various areas in Asia, Latin America, and the Middle East ( 10 ).

Notably, in the United States, S. flexneri was the predominant serotype in the early 1960s, but it was supplanted by S. sonnei between 1964 and 1968,  the cause of which remains unknown ( 10 ).

The increasing prevalence of S. sonnei in developing countries could potentially be attributed to improvements in water quality and sanitation practices. These improvements might limit the passive immunization typically conferred by P. shigelloides, which is commonly found in contaminated water sources. Furthermore, the amoeba Acanthamoeba castellani serves as a reservoir for S. sonnei, enabling its persistence even in highly chlorinated environments where S. flexneri struggles to thrive. Additionally, S. sonnei demonstrates a greater propensity for acquiring resistance compared to S. flexneri, giving it a competitive edge, particularly in regions with limited antimicrobial usage ( 11 ).

Shigella spp. can easily acquire and spread antimicrobial resistance genes, as in recent years, MDR-positive Shigella spp. have been reported abundantly  throughout the world. In this study, all isolates were resistant to at least three classes of antibiotics and were considered MDR. Also, 54% of isolates produced ESBL, which could be a serious threat to public health. The frequency of ESBL-producing Shigella isolates was reported to be 7.5% by Ranjbar et al., in 2013 ( 12 ) in Tehran, Iran, and the results of this study, in comparison to their findings, represented a significant increase in ESBL-producing Shigella isolates in other regions of Iran. In a cross-sectional study in Ardabil, Iran, from 2019 to 2020, 10.2% of Shigella species were ESBL positive, and various beta-lactamase genes including bla_CTX-M and bla_TEM were found among them ( 13 ). In another study in Tehran, Iran from 2015 to 2017 bla_CTX-M-15 (10.7 %), bla_SHV (28 %), and bla_TEM (21.3 %) were reported among Shigella isolates ( 14 ). In the present study, bla_TEM and bla_CTX-M were the common ESBL genes among the isolates, with frequencies of 84 (40%) and 93 (44%), respectively, which is similar to findings in Argentina, Turkey, Lebanon, China, Korea, and Japan, where bla_TEM and bla_CTX were as the predominant ESBL genes in Shigella isolates ( 15 - 17 ).

This study revealed that more than 50% of the Shigella isolates exhibited resistance to ampicillin, tetracycline, trimethoprim-sulfamethoxazole, streptomycin, chloramphenicol, cefotaxime, and ceftriaxone. Many reports have highlighted a significant prevalence of resistance to ampicillin and trimethoprim-sulfamethoxazole among Shigella isolates, and based on these reports, trimethoprim-sulfamethoxazole and ampicillin are not appropriate choices for the treatment of shigellosis ( 18 , 19 ).

A high prevalence of resistance to nalidixic acid, trimethoprim/sulfamethoxazole, and ampicillin was reported in Shigella isolates among pediatric patients in different regions of Iran ( 14 ). Therefore, findings in different regions in Iran, similar to our results, showed that the rate of resistance to ampicillin, nalidixic acid, and trimethoprim/sulfamethoxazole is high.

In contrast to the findings by Xing et al. in China ( 18 ), in this study, resistance to nalidixic acid (31%) was low. This difference may be to the limited or nonexistent use of this drug in the treatment of shigellosis and other gastrointestinal tract infections in Iran. Gu et al., ( 20 ) demonstrated a significant increase in ciprofloxacin resistance in Asia and African countries over 12 years, whereas resistance to this antibiotic and third-generation cephalosporins was reported to be below 1% in the United States and some European countries. Ceftriaxone is presently the primary treatment for shigellosis in hospitalized patients. However, the irregular use of antibiotics has contributed to the development of resistant strains. Resistance to third-generation cephalosporins, particularly ceftriaxone, has been notably high in countries like Vietnam, China, and Iran. Despite reports of increasing antibiotic resistance in Shigella spp, the high resistance rate (49%) to ceftriaxone in this study is particularly noteworthy.

In a study conducted by Jafari et al., in 2009 ( 21 ) in Tehran, Iran, more than 90% of Shigella isolates were found to be susceptible to ceftriaxone, ceftazidime, cefepime, and ciprofloxacin. Conversely, Mostafavi et al., ( 22 ) reported a very high level of resistance among Shigella serotypes to trimethoprim-sulfamethoxazole, ampicillin, and third-generation cephalosporins. Furthermore, studies conducted in Iran over the past 20 years have consistently revealed a high level of resistance to trimethoprim-sulfamethoxazole and ampicillin ( 22 - 25 ).

In this study, a significant relationship was observed between all the S. sonnei strains and resistance to trimethoprim-sulfamethoxazole (P ≤ 0.5). Additionally, in the current study, S. boydii, S. flexneri, and S. dysenteriae strains exhibited a significant relationship with resistance to trimethoprim-sulfamethoxazole, ampicillin, and streptomycin (P ≤ 0.05). This may indicate a clonal spread of these strains due to the horizontal transfer of plasmids carrying multiple resistance genes in Iran. This highlights the key role of horizontal transfer of multidrug resistance genes associated with endemic plasmids. Overall, the low resistance to ciprofloxacin (6%) in this study suggests that fluoroquinolones remain effective for treating shigellosis in different regions of Iran. However, due to the limitations of fluoroquinolone prescription in children because of their side effects, some cephalosporins are often used as an alternative treatment for shigellosis.

The PBRT method is a practical tool for evaluating the genetic relatedness of bacterial isolate in epidemiological studies. In this study, plasmid profiling showed that 72.8% of isolates harbored I1-Iγ replicon type as the most abundant replicon type. I1-Iγ replicons are limited to Enterobacteria hosts carrying  the bla_CTX-M, bla_SHV, bla_CMY, bla_TEM genes, integrons and resistance genes to arsenic, tetracycline and streptomycin.

In the study by Ruekit et al.,( 26 ) conducted in India on 29 S. sonnei isolates, only B/O (34.4%) and I1-Iγ (13.7%) replicon types were reported, while in this study, I1-Iγ and B/O in were reported in 72.8% and 1.4% of isolates, respectively. Due to the geographical distribution and species diversity of isolates in this study, clonal dissemination of Shigella isolates carrying the I1-Iγ replicon is unlikely. Also, in the present study, most ESBL isolates carried the I1-Iγ replicon, so there may be a co-relationship between these two factors. The results of this study suggests that the I1-Iγ replicon is likely more compatible with ESBL-positive Shigella isolates than other plasmid replicon types.

In the present study, isolates harboring multi-replicon types were observed abundantly, such that among plasmid-carrying isolates, 59.3% harbored at least three replicons simultaneously. Also, two isolates, both belonging to S. soneii from Kerman, carried eight replicon types. The presence of multiple replicons among the isolates is one of the main factors in facilitating genetic exchanges, therefore, while increasing the chance of horizontal gene transfer among isolates (a broad host range), it also increases the possibility of genetic recombination between different plasmids within each isolate, which can drive the evolution and diversity of these bacteria, potentially leading to new pathogenicity and resistance profile.

In conclusion, the study sheds light on the persistent challenges posed by shigellosis to global public health. Despite advancements in hygiene practices, the prevalence and population composition of Shigella species remain largely unchanged. The epidemiological transition from S. flexneri to S. sonnei observed in various regions, underscores the dynamic nature of this infectious disease. Antimicrobial resistance among Shigella spp. poses a significant threat, with multi-drug resistance and ESBL-producing isolates increasing worldwide, which may reduce treatment options.

Notably, the high resistance rate to ceftriaxone in Iran underscores the urgent need for prudent antibiotic stewardship. Additionally, this study highlights the possible role of plasmid replicon types in the development and dissemination of resistance genes among Shigella isolates, and the spread of the I1-Iγ replicon, which is usually associated with ESBL genes, underscores the importance of understanding plasmid dynamics in combating antimicrobial resistance. However, despite the challenges posed by antimicrobial resistance, some antibiotics like ciprofloxacin remain effective for treating shigellosis, although its use is limited, especially in pediatric patients. Ongoing surveillance and appropriate use of antibiotic agents are essential for managing shigellosis caused by drug-resistant isolates.

Acknowledgment

The authors are grateful Department of Medical Microbiology (Bacteriology and Virology), Faculty of Medicine, Kerman University of Medical Sciences, Kerman, Iran.

Authors' Contribution

Study concept and design: SA. AM, D. K-N.

Acquisition of data: M. M, H. HN.

Analysis and interpretation of data: D. K-N, P. M, SA. AM.

Drafting of the manuscript: D. K-N, P. M, SA. AM.

Critical revision of the manuscript for important intellectual content: D. K-N, P. M.

Statistical analysis: D. K-N, P. M.

Administrative, technical, and material support: D. K-N.

Ethics

The present study with Reg. No. 97000972 was approved by the ethical committee of Kerman University of Medical Sciences, Kerman, Iran. The ethics approval code is IR.KMU.REC.1398.199.

Conflict of Interest

All authors of this manuscript have no conflicts of interest to disclose.

Funding

This study was supported by the Kerman University of Medical Sciences science grant number 97000972.

Data Availability

The datasets used or analyzed during the study are available on reasonable requests from the corresponding author (Email: d.kalantar@kmu.ac.ir).

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