Iranian Journal of Medical Microbiology

2.1. Sampling and Identification of Bacteria
This descriptive cross-sectional study involved the utilization of 269 E. coli isolates obtained from urine samples of hospitalized patients in Rasht, a city in northern Iran. The isolates underwent a series of laboratory procedures. Initially, Gram staining was performed, followed by inoculation on blood agar and EMB agar. The plates were then incubated at 37°C for 48 hours. Standard biochemical tests, including oxidase, citrate, urea, triple sugar iron, SIM, lysine decarboxylase, methyl red, and Voges-Proskauer tests from Merck Co., Darmstadt, Germany, were employed to identify the grown colonies as E. coli. To serve as a control, E. coli PTCC No. 1399 (ATCC 25922) was obtained from the Iran Research Organization for Science and Technology (IROST).
2.2. Phenotypic Confirmatory Disc Diffusion Test (PCDDT) For ESBL Detection
Each of the test isolates was individually inoculated on Mueller Hinton agar at a final concentration of 0.5 McFarland and then incubated at 37°C for 24 hours. Ceftazidime (30 μg) and ceftazidime-clavulanic acid (30 μg/10 μg) disks were employed in this test. An increase in the zone diameter of ≥5 mm in the presence of clavulanic acid, compared to ceftazidime alone, was considered indicative of ESBL production. (10).
2.3. Antimicrobial Susceptibility Testing
The ESBL-producing isolates were subjected to antimicrobial susceptibility testing using the Kirby-Bauer disk diffusion method. The following antibiotics were tested: Cefotaxime (30), Ampicillin (10), Ciprofloxacin (5), Cefoxitin (30), Aztreonam (30), Amoxicillin-clavulanate (30), Tetracycline (30), Ofloxacin (5), Cephalothin (30), Gentamicin (120), Nalidixic acid (30), Cotrimoxazole (25), Cefixime (5), Nitrofurantoin (300), Imipenem (10), Ceftriaxone (30), and Ceftazidime (300). The antibiotics used in the study were provided by Mast Co., Merseyside, UK.
For the testing, a bacterial suspension containing 1.5 × 10⁸ CFU/mL (0.5 McFarland standard) of each strain was prepared, and 0.1 mL of the suspension was cultured separately on Mueller-Hinton agar. The antibiotic-containing disks were then placed on the surface of the medium. To determine antibiotic susceptibility, E. coli PTCC No. 1399 (ATCC 25922) was used as a control.
Following incubation, the zones of inhibition surrounding the antibiotic disks were measured and compared to the standard guidelines for each antibiotic, as outlined in the Clinical and Laboratory Standards Institute (CLSI-2020-M100-S30) (11). This comparison was done against the bacteria being investigated.
2.4. Plasmid Extraction
The plasmid DNA from all ESBL-producing E. coli isolates was extracted from fresh colonies using the Gene JET Plasmid Miniprep Kit (Fermentas, Lithuania), following the instructions provided by the manufacturer. The DNA concentration of each extracted plasmid DNA was determined using Nanodrop. The extracted DNA was then stored at -20 °C and used as a template for subsequent amplifications.
2.5. PCR Amplification, Electrophoresis and S Sequencing
The PCR amplification was conducted using specific primers listed in Table 1, which were synthesized by Metabion Company (Germany). The PCR reaction mixture consisted of 10 μL of Master Mix (PCRBIO Taq Mix Red 2X-1ml-AgG), 8.5 μL of ddH₂O, 0.5 μL of each primer (10 pmol), and 0.5 μL (50-100 nmole) of plasmid DNA as the template. The amplification reaction was carried out according to the conditions outlined in Table 2.
All PCR products were subjected to electrophoresis on a 1.5% agarose gel containing SYBR Safe. The molecular weight of the observed bands was determined using a DNA size marker under a UV lamp. The PCR products were then sent to Topazgene Co., Alborz, Iran, for sequencing. Sequence analyses and comparisons were performed using programs available on the NCBI server [http://www.ncbi.nlm.nih.gov].
2.6. Statistical Analysis
The frequency of resistance and housekeeping genes were analyzed using SPSS^TM software, 26.0 (IBM Corp., USA).

Table 2. Gene amplification program in PCR

When it comes to treating infections with antibiotics, it is crucial to consider local experience regarding susceptibility and resistance patterns. This is because the susceptibility of microorganisms to antibiotics can vary over time and in different geographical regions. In this particular investigation, every isolate demonstrated multidrug resistance (MDR). For empirically treating urinary tract infections caused by ESBL-producing strains, the most effective antibiotics were found to be imipenem and nitrofurantoin.
It is important to note that cefotaxime, ampicillin, amoxicillin-clavulanate, cephalothin, cefixime, and ceftriaxone exhibited high rates of resistance and are not recommended for the treatment or prevention of these strains in the Guilan region. The high frequency of virulence factors and resistance genes observed in this study is significant as it can lead to increased pathogenicity and treatment failure.
Our findings align with the study carried out by Negeri et al. in 2021. In their research, they examined the molecular epidemiology of ESBL genes and the antibiotic resistance profile of 204 clinical E. coli isolates in Ethiopia. According to their data, all of the isolates demonstrated multidrug resistance (MDR). Furthermore, out of the 189 bacteria that had ESBL genes, all except one had CTX-M β-lactamases (20).
In a separate investigation, Abdelrahim et al. (2021) demonstrated that O25-ST131 isolates exhibited a significantly higher frequency of ESBLs and MDR occurrence. These isolates also showed antibiotic resistance to a majority of antibiotic classes, in contrast to non-O25-ST131 isolates (21).
Rasoulinasab et al. (2021) collected 215 E. coli isolates from urine samples obtained from women experiencing symptomatic urinary tract infections (UTIs) at referral university hospitals in Tehran, Iran. They specifically investigated ciprofloxacin-resistant ESBL-producing (CIPR/ESBL+) E. coli. The study reported a prevalence of 82% for the blaCTX-M-15 gene and resistance rates of 70% for ampicillin-sulbactam, 97% for aztreonam, and 61% for gentamicin (22).
In our study, we found a lower resistance rate for gentamicin (32.7%) and higher resistance rates for ciprofloxacin (78.5%), ampicillin (100.0%), and aztreonam (83.2%) when compared to the findings of Rasoulinasab's study. Furthermore, the prevalence of CTX-M-15 in our study was 86.9%.
Namaei et al. (2017) reported on the dissemination of the O25b/ST131 clone that produces CTX-M-15 among E. coli isolates. This study provided the first documented frequency of O25b/ST131 E. coli producing CTX-M-15 in Birjand, Iran. The authors observed a comparatively high prevalence of O25b/ST131 E. coli, which exhibited significant levels of virulence factors and antibiotic resistance. Additionally, the majority of these isolates carried the CTX-M-15 gene and were capable of producing ESBLs. These findings highlighted the spread of ST131 E. coli as a significant drug-resistant bacterium and a new threat to public health (23).
In our study, we found that the abundances of ST131-H30, O25b-ST131, ST131-H30-Rx, and O16-ST131 were 64.5%, 63.5%, 60.7%, and 50.5%, respectively, based on our findings.
In their study, Bulut et al. (2021) investigated the prevalence of high-risk E. coli ST131 clones among ESBL-positive E. coli isolates in Turkey. The study reported a high rate of ST131 clones within the population. The notable resistance observed in ST131 isolates against ciprofloxacin highlights the significant implications of the dissemination of these high-risk clones for the development of resistance (24). These findings align with the results of our study.
Other studies have documented the presence of antibiotic resistance and virulence genes in uropathogenic E. coli. Dziri et al. (2020) conducted research on the uropathogenic O25b-ST131 clone, determining its frequency and identifying the molecular pathways responsible for cephalosporin resistance in clinical E. coli isolates. During the period spanning from April 2015 to August 2016, a total of 42 E. coli strains resistant to cephalosporins, which were linked to urinary tract infections, were gathered from the provincial hospital situated on an island in southeast Tunisia. Most of their isolates were found to contain the CTX-M gene, which was detected through molecular screening of genes encoding β-lactamases using PCR and sequencing. This investigation marks the first report of O25b-ST131 E. coli producing CTX-M-14 on the island of Tunisia (25). Demirci-Duarte et al. (2020) conducted a study in Turkey to examine the frequency of the ST131 clone and the presence of CTX-M in 299 urine isolates. The researchers also analyzed the antimicrobial susceptibility. The study found that the ST131 clone was detected in 31.7% of the isolates that were resistant to ciprofloxacin, accounting for 34.7% of all isolates. Furthermore, it was observed that 81.8% of the ST131 clone belonged to the H30-R subclone, while 66.7% belonged to the H30-Rx subclone. However, the study revealed that 57.6% of isolates from the ST131 clone had CTX-M, in comparison to 62.9% in the H30-R subclone and 68.2% in the H30-Rx subclone. These findings suggest that the occurrence of CTX-M-positive ST131 clones is relatively high (26). In their study, Hojabri et al. (2017) assessed the prevalence of the O25b/O16 subgroups of the ST131 clone and the H30/H30-Rx lineages in E. coli isolates that cause extraintestinal infections. The aim was to understand their role in the dissemination of antimicrobial resistance. Additionally, PCR was used to evaluate the genetic diversity and virulence properties of the ST131 isolates. The findings of this study confirm the increasing presence of the H30 subclone in our region and suggest that this particular subclone is primarily responsible for the successful expansion of the ST131 clonal group.
Despite the lower frequency of the O16-ST131 clonal subgroup compared to O25b-ST131, it showed higher rates of resistance to most antibiotics. This observation highlights the significance of the O16-ST131 subgroup in the dissemination of multidrug-resistant E. coli (27). Salehi et al. (2023) conducted a study in Mashhad, Iran, to determine the pattern of antibiotic resistance and the frequency of specific genes in E. coli isolates. The results of their research indicated that a significant portion of the isolates exhibited resistance to antibiotics and were identified as E. coli (28). Several studies conducted in various provinces of Iran have demonstrated the presence of multidrug-resistant extended-spectrum beta-lactamase (MDR-ESBLs)-producing E. coli (29-33). The findings from these studies corroborate the current study's findings regarding the prevalence of virulence genes and antibiotic resistance in ExPEC strains responsible for urinary tract infections (UTIs). However, it is worth noting that the results of these studies vary, which could be attributed to differences in sample characteristics, geographical locations, sample sizes, and other factors.

 

When comparing these data with those from other countries, significant variations in the susceptibility and resistance characteristics of the bacteria become apparent. As a result, the choice of antibiotics for empirical therapy should be guided by the resistance patterns of the uropathogens specific to each city. Moreover, it can be inferred that the increase in virulence factors has contributed to the emergence of multidrug-resistant extended-spectrum beta-lactamase (MDR-ESBLs)-producing ExPEC strains.

 

We gratefully acknowledge the support of the Microbiology Department, Faculty of Medicine, Guilan University of Medical Sciences, Rasht, Iran. The authors acknowledge Dr. Hadi Sedigh for his kind cooperation.
 

Ethics Approval

The study procedures adhered to the ethical guidelines outlined in the Declaration of Helsinki. It is important to note that the test microorganisms were obtained from Dr. Mojtahedi's previous research. However, to ensure the authenticity of each E. coli sample, microscopic and biochemical tests were performed. All methods were conducted in accordance with applicable guidelines and regulations.

This paper was derived from the Ph.D. thesis of the first author, with a registration dissertation code of 157482802614479162249610. The proposal was approved by the North Tehran Branch of the Islamic Azad University in Tehran, Iran.

 

Authors’ Contribution

Conceptualization, methodology, resources, review, and editing: All authors; Investigation and original draft: Samaneh Kazemi; Supervision: Ali Mojtahedi & Farzaneh Hosseini.

Conflicts of Interest

The authors declared no conflict of interest.