Quinolone Resistance and Virulence Genes Prevalence of Uropathogenic Escherichia coli Isolated from Kidney Transplant Recipients with Urinary Tract Infections in Tehran, Iran

Introduction

Kidney transplantation is the final treatment option for individuals with end-stage kidney disease, significantly enhancing their survival rates and quality of life. However, infections that occur post-transplant pose a significant challenge, potentially leading to serious complications and increased mortality.¹ Urinary tract infections (UTIs) are especially concerning because they are associated with an increased risk of graft dysfunction and higher healthcare costs.² Various factors increase the risk of UTIs in kidney transplant recipients, such as pre-transplant renal impairment, diabetes mellitus, older age, and a history of recurrent UTIs.¹ UTIs can lead to sepsis, and recent data indicate that even a single UTI episode may negatively affect graft function, in addition to the risk of progressing to septicemia.^3,4

Among the bacteria responsible for UTIs, uropathogenic Escherichia coli (UPEC) is the most common cause of UTIs, accounting for 90% of community-acquired and 50% of hospital-acquired infections.⁵ This bacterium has developed several virulence factors, such as fimH (type 1 fimbriae), hlyA (hemolysin), and ompT (outer membrane protein T). These factors play crucial roles in adhesion, invasion, and resistance to the host’s immune defenses, contributing to the severity of UTIs.^6,7 Due to the diverse phylogenetic backgrounds of E. coli, the presence and expression of these virulence factors can vary significantly.⁸

The World Health Organization (WHO) has classified fluoroquinolones as a critical group of antibiotics frequently prescribed to kidney transplant patients who have been infected with E. coli. However, increased antibiotic resistance has restricted their use.^9–11 Resistance to quinolones in E. coli isolates occurs through the protection of DNA gyrase and topoisomerase IV from the action of quinolone antibiotics. This resistance is mediated by plasmid-borne quinolone resistance genes such as qnrA and qnrB.¹¹ Additionally, the aminoglycoside-modifying enzyme, encoded by the aac(6′)-Ib gene, contributes to this resistance by acetylating fluoroquinolones, which reduces susceptibility to these antibiotics.¹²

Given the infections that occur as a result of transplantation cause high mortality, further research is essential to understand the contributing factors associated with antibiotic resistance development and severity of UTIs in transplant patients.⁸ Accordingly, this study aims to investigate the prevalence of key virulence genes and quinolone-associated resistance genes in E. coli isolates from patients with post-kidney transplant UTIs.

Methods

In this study, all kidneys were donated voluntarily with written informed consent, and that these were conducted in accordance with the Declaration of Istanbul.

Urine Collection

Also, urine collection was done as a part of the routine hospital laboratory procedure for all patients with UTIs signs. All urine samples were collected in sterile containers based on mid-stream clean-catch method.

Bacterial Isolation and Identification

This study was conducted using 50 non-duplicate E. coli isolates obtained from kidney transplant patients suffering from UTIs referred to Yekta and Gholhak laboratories and Labbafi Nezhad Hospital in Tehran, Iran, from February 2022 to May 2024. In this study, urine samples were collected in sterile containers. The urine samples were then cultured on three media, including blood Agar, EMB Agar, and MacConkey Agar. Subsequently, biochemical tests were performed on Gram-negative isolates suspected to be Enterobacteriaceae. The isolates, which had been detected as E. coli, were later transferred to the microbiology research laboratory at Shahid Beheshti University of Medical Sciences, Tehran, Iran. This study was approved by the ethics committee of Shahid Beheshti University of Medical Sciences (ethical approval code: IR.SBMU.RETECH.REC.1403.533). The isolates were confirmed using Gram staining and standard biochemical tests, including Catalase, Oxidase, IMViC (Indole, Methyl Red, Voges-Proskauer, and Citrate Utilization), Triple Sugar Iron Agar (TSI), Urease, Motility and Oxidative Fermentation tests on pure isolates on Blood Agar and MacConkey Agar.¹³ They were stored in a 10% glycerol and TSB solution at −70°C for further evaluation.

Antimicrobial Susceptibility Test

Antimicrobial susceptibility testing was conducted using the Kirby–Bauer disc diffusion method according to Clinical Laboratory and Standards Institute (CLSI) guidelines,¹⁴ and E. coli ATCC 25922 was used as a control strain. The antibiotics used in this study included ampicillin (10 μg), ampicillin-sulbactam (10/10 μg), amoxicillin-clavlunate (20/10 μg), cefotaxime (30 µg), cefoxitine (30 µg), cefepime (30 µg), cefazolin (30 µg), imipenem (10 μg), meropenem (10 μg), ertapenem (10 μg), doripenem (10 μg), amikacin (30 μg), tobramycin (10 μg), gentamicin (10 μg), piperacillin/tazobactam (100/10 μg), ciprofloxacin (5 μg), fosfomycin (200 μg), nitrofurantoin (300 μg), trimethoprim (5 μg), cefpodoxime (10 μg) purchased from Mast group (Merseyside, UK) and Rosco (Taastrup, Denmark) company.

DNA Extraction

Bacterial DNA was extracted using the boiling method. Briefly, pure E. coli colonies were harvested from overnight cultures and washed with sterile distilled water. The cell pellet was re-suspended in sterile distilled water, vortexed, and then boiled at 100°C for 10 minutes. The lysate was immediately cooled on ice for 5 minutes and centrifuged to remove cellular debris.¹⁵ The supernatant containing genomic DNA was collected and stored at −70°C for further molecular investigation.

Detection of Virulence Factors, and Fluoroquinolones Resistant Genes

As described in Table 1, PCR with specific primers was utilized to screen for hlyA, fimH, ompT, aac(6´)Ib, qnrA, and qnrB genes. The PCR reaction was carried out in a total volume of 25 μL that contained 1.5X Taq PCR Master Mix, 1 µM of each primer, and 2 μL of template DNA. The PCR products were subjected to electrophoresis on a 1.5% agarose gel. In this study, three E. coli isolates were used as controls, including ATCC 25922, an E. coli strain that carries both the hlyA and fimH genes and dedicated by Dr. Shiva MirKalantari (Iran University of Medical Sciences) along an qnrA+ E. coli isolate from our previous research.¹⁵

Table 1 Primer Sequences, Product Sizes, and Annealing Temperatures for Quinolone Resistance and Virulence Genes in Clinical Isolates of E. coli

Sequencing

Sanger sequencing was conducted by the Metabion company to confirm the presence of ompT, aac(6′)Ib, and qnrB genes in a clinical isolate, which harbored each of these genes. Further nucleotide analyses were performed using Chromas 1.45 software and BLAST in NCBI.

Statistical Analysis

Descriptive statistics were utilized in this study using GraphPad Prism software version 9.4.1 to assess the frequency of antibiotic resistance and virulence genes. Prevalence estimates are provided as n (%) with 95% confidence intervals (CIs) using the Wilson score method. No comparative analyses were performed.

Results

Bacterial Identification and Antimicrobial Susceptibility Results

All E. coli colonies were identified and confirmed after visualization of Gram-negative bacilli, Oxidase (-), TSI A/A gas (+), H 2 S (-), IMViC (+ + - -), Urease (-), and Motility (+). Antimicrobial susceptibility testing of isolates which have been interpreted according to the CLSI guideline was displayed that the highest resistance rate was for ampicillin (94%, CI: 83.5–98.0), followed by cefazolin (88%, CI: 76.2–94.4), fosfomycin (86%, CI: 73.4–93.1), cefotaxime (80%, CI: 66.8–88.9), cefpodoxime (80%, CI: 66.8–88.9), meropenem (72%, CI: 58.1–82.5), trimethoprim/sulfamethoxazole (70%, CI: 56.2–80.9), cefepime (70%, CI: 56.2–80.9), ampicillin-sulbactam (64%, CI: 50.1–75-9), ciprofloxacin (62%, CI: 48.2–74.1), amoxicillin-clavulanate (54%, CI: 40.4–67.0), piperacillin-tazobactam (54%, CI: 40.4–67.0), cefoxitin (48%, CI: 34.8–61.5), tobramycin (44%, CI: 31.2–57.7), gentamicin (34%, CI: 22.4–47.8), amikacin (28%, CI: 17.5–41.7), ertapenem (20%, CI: 11.2–33.0), nitrofurantoin (20%, CI: 11.2–33.0), imipenem (18%, CI: 9.8–30.8), doripenem (1%, CI 0.0–7.1), as illustrated in Figure 1.

Figure 1 Antibiotic resistance rate in E. coli isolates from kidney transplantation with urinary tract infections. Resistance was assessed by disk diffusion method. Abbreviations: AMP, Ampicillin; AMC, Amoxiclav; AMP/SUL, Ampicillin/Sulbactam; PIP/TAZ, Piperacillin/Tazobactam; CFZ, Cefazolin; FEP, Cefepime (fourth-generation cephalosporisn); CTX, Cefotaxime; FOX, Cefoxitin; CPD, Cefpodoxime; DOR, Doripenem; ETP, Ertapenem; MEM, Meropenem; IMP, Imipenem; GEN, Gentamicin; TOB, Tobramycin; AMK, Amikacin; CIP, Ciprofloxacin; TMP, Trimethoprim; NIT, Nitrofurantoin; FOS, Fosfomycin.

Identification of Resistance and Virulence Genes by PCR

PCR results demonstrated that among E. coli isolates from kidney transplant recipients with UTI, fimH had the highest frequency (82%, CI: 69.2–90.2), followed by ompT (40%, CI:27.6–53.8), aac (6´) Ib (30%, CI: 19.1–43.8), qnrA (18%, CI: 9.8–30.8), hlyA (10%, CI: 4.3–21.4), and qnrB (8%, CI: 3.2–18.8), which were at a lower level (Figure 2). Representative PCR-gel electrophoresis images are provided in a Supplementary Figure 1.

Figure 2 Frequency of quinolone resistance and virulence genes detected by PCR among E. coli isolates from kidney transplantation with urinary tract infections.

The simultaneous presence of invasive and resistance genes was observed in several isolates. Notably, 28% (CI: 17.5–41.7) of the isolates carried both aac (6´) Ib and fimH, 20% (CI: 11.2–33.0) had aac (6´) Ib and ompT, and 6% (CI: 2.1–16.2) demonstrated aac (6´) Ib and hlyA. Moreover, 16% (CI: 8.3–28.5) of E. coli isolates harbored both qnrA and fimH, while 4% (CI: 1.1–13.5) had qnrA and ompT. None of the isolates concurrently exhibited qnrA and hlyA, qnrB and hlyA (CI: 0.0–7.1). Additionally, 4% (CI: 1.1–13.5) of isolates showed the presence of qnrB and fimH, and 4% (CI: 1.1–13.5) co-harbored qnrB and ompT (Figure 3).

Figure 3 Co-occurrence of resistance and virulence genes in E. coli isolates from kidney transplantation with urinary tract infections.

Discussion

Kidney transplant recipients are at increased risk of UTIs due to the long-term use of immunosuppressive drugs. Among the various bacteria, E. coli is the most common pathogen associated with these infections and is often linked to multidrug resistance (MDR).²² In the current study, the resistance rates to ampicillin and ampicillin/sulbactam within the β-lactam class were higher than those for other antibiotics in this group. Additionally, the resistance rate to ciprofloxacin, a fluoroquinolone, was notably high, exceeding 60%. These findings are consistent with previous research.^23,24 However, other epidemiological investigations reported a lower frequency of fluoroquinolone resistance.^25,26 The discrepancies between these findings may be attributed to variations in geographical regions, antibiotic prescribing patterns, and infection control policies in their respective countries.

In addition to epidemiological factors, genetic mechanisms also play a critical role in the development of fluoroquinolone resistance patterns. The emergence of antibiotic resistance can be associated with genes that confer resistance, including plasmid-mediated determinants that specifically affect fluoroquinolone susceptibility.²⁷

The findings revealed that the prevalence of each of the qnrA, qnrB, and aac (6′)-Ib genes stood at 18%, 8%, and 30%, respectively. Raheem et al reported a pattern comparable to that observed in the present study. Their research highlighted that the qnr gene was detected in 18% of UPEC isolates.²⁸ Similarly, other studies have reported a low prevalence of qnr genes in clinical isolates.^29,30 Given that 30% of the isolated strains harbored the aac (6′)-Ib gene, it is likely that this gene can contribute to the development of fluoroquinolone resistance.³¹ In the similar investigation conducted by Esmaeel et al, the aac (6′)-Ib gene was identified as the most common fluoroquinolone resistance gene. However, the prevalence of qnr genes was found to be higher than what we reported.³² Additionally, Badamchi et al documented that the aac(6′)-Ib gene was present in 24% of E. coli isolates from patients in Iran.³³

Research indicates that antibiotic-resistant genes exhibit varying prevalence across different studies and geographical regions. This variability can be linked to antibiotic usage patterns and different consumption regimes.³⁴ In addition to antimicrobial resistance, bacterial virulence factors significantly influence the severity and clinical outcome of urinary tract infections.³⁵

Various virulence factors contribute to the pathogenicity of E. coli, which can be detected through PCR.³⁶ The gene coding for FimH adhesion is recognized as one of the consequential factors in the virulence of E. coli in UTIs, facilitating pathogenicity through the colonization, invasion, and persistence of UPEC in the bladder.³⁷ Our findings indicate a higher frequency of fimH gene than other examined genes, suggesting that this gene plays a crucial role in E. coli infections causing UTI. The distribution of this gene in our strains aligns with previously published data.^38,39 However, other studies have noted a higher percentage of fimH PCR-positive strains in patients with UTIs.^40–42 This discrepancy can be explained by the occurrence of mutations and the failure to detect them.³⁹ In this investigation, the frequencies of the hlyA and ompT genes were 10% and 40%, respectively. However, the prevalence of these virulence factors varies across other studies, which differences in sample sources, geographical regions, and host clinical conditions can explain this discrepancy.^43,44 Furthermore, our findings indicated a considerable number of isolates carrying both invasive and resistance genes simultaneously, which aligns with previous studies.^45,46 Isolates that concurrently harbored both types of genes may cause more severe infections or exhibit a poor response to treatment. Additionally, the co-existence of invasive and resistance genes on a plasmid could pose a heightened threat to E. coli infection control, as they can be co-transferred and spread to other isolates.

Conclusion

The current study reveals significant levels of antibiotic resistance and the distribution of virulence genes in E. coli isolates from kidney transplant recipients with UTIs. To the best of our knowledge, the current study represents the first report of the simultaneous occurrence of plasmid-mediated quinolone resistance and invasive genes in E. coli isolates from UTIs in kidney transplant patients, which may be associated with poor therapeutic outcomes. High phenotypic and molecular resistance rates to therapeutic antibiotics, such as ciprofloxacin, increase the risk of treatment failure in E. coli UTI infections. The high prevalence of fimH, which is associated with bacterial adhesion, colonization, and persistence of E. coli in the urinary tract, also emphasizes the role of this gene in the development of E. coli infection in UTI patients. Collectively, these findings highlight the critical need to apply both phenotypic antimicrobial susceptibility testing and molecular screening for antibiotic resistance, along with analysis of virulence genes, to develop effective treatment strategies and prevent E. coli infections in patients with UTIs. Further multicenter, longitudinal studies are required to examine the clinical impact of these genetic profiles across a broader range of clinical isolates to determine treatment outcomes and inform evidence-based therapeutic guidelines.