Molecular Investigation and Confirmation of Clinical Isolates of Escherichia coli Urinary Infections and Evaluation of Frequency of qnrA Quinolone Resistance Gene by PCR Method

¹ Infection Disease Research Center, Hamadan University of Medical Sciences, Hamadan, Iran

² Infectious Ophthalmologic Center, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran

³ Comprehensive Research Laboratory, Hamadan University of Medical Sciences, Hamadan, Iran

⁴ School of Medicine, Hamadan University of Medical Sciences, Hamadan, Iran

*Corresponding author: Elham Abdoli, Infection Disease Research Center, Hamadan University of Medical Sciences, Hamadan, Iran. Email: eabdoli8387@yahoo.com

EXTENDED ABSTRACT

Background

Escherichia coli is a Gram-negative facultative anaerobic bacillus of the Enterobacteriaceae family, and many strains behave as opportunistic pathogens [1]. Extraintestinal pathogenic E. coli can cause substantial human disease, and the source article notes that E. coli accounts for approximately 90% of urinary tract infections (UTIs) in young women [2, 3], 80-90% of UTIs in outpatients, and 30-50% of UTIs in hospitalized patients [4]. Approximately 150 million UTI episodes are diagnosed worldwide each year [5, 6], and uropathogenic E. coli may colonize the urinary tract particularly in the presence of predisposing conditions such as urinary stones, immune dysfunction, urinary surgery, tumors, pregnancy, diabetes, tuberculosis, or urinary instrumentation [12]. Inappropriate antibiotic therapy has contributed to increasing antimicrobial resistance among urinary E. coli isolates, making local knowledge of resistance patterns and mechanisms important for effective treatment selection [13]. Misuse of antibiotics in humans and extensive use in food-producing animals are among the factors associated with antimicrobial resistance [14, 15], which has become a major challenge for prevention and treatment of infectious diseases [16]. E. coli can acquire and disseminate resistance determinants through mobile genetic elements, including transposons, bacteriophages, and plasmids [17]. Quinolones are broad-spectrum agents commonly used against UTIs. Resistance may arise through chromosomal changes involving DNA gyrase and topoisomerase IV or through plasmid-mediated mechanisms, including the Qnr pentapeptide-repeat proteins; the qnr family includes qnrA, qnrB, qnrC, qnrD, and qnrS [14]. The present study therefore aimed to phenotypically and molecularly confirm clinical E. coli isolates recovered from UTIs in hospitalized patients, determine their quinolone resistance profile, and evaluate the frequency of the qnrA resistance gene.

Methods

This cross-sectional study was conducted over a 9-month period in 1402 of the Iranian calendar among hospitalized patients at Sina Hospital in Hamadan. Based on the estimated sample size, 100 E. coli isolates recovered from urine samples of patients with UTIs were obtained through the Comprehensive Research Laboratory of Hamadan University of Medical Sciences. Following Gram staining and recognition of Gram-negative bacteria, oxidase and catalase testing was performed. Differential and biochemical media, including phenylalanine deaminase, lysine iron agar, Simmons citrate, methyl red-Voges-Proskauer, sulfide-indole-motility, triple sugar iron, and urease media, were used for phenotypic identification. Isolates were streaked on MacConkey agar, incubated for 18-24 hours at 37 degrees C, and suspicious pink colonies were subcultured on eosin methylene blue agar for confirmation and purification [2]. Suspected colonies were further evaluated by IMViC and related biochemical reactions. Antimicrobial susceptibility testing was performed by the Kirby-Bauer disk diffusion method on Mueller-Hinton agar and interpreted according to Clinical and Laboratory Standards Institute criteria [18]. The evaluated quinolone agents were ciprofloxacin, levofloxacin, and nalidixic acid; E. coli ATCC 25922 was used for quality control. Genomic DNA was extracted from the isolates by the boiling method, quantified using a NanoDrop spectrophotometer, and evaluated for quality by absorbance ratios and electrophoresis on 2% agarose gel [4]. Molecular confirmation of E. coli was performed using species-specific 16S rRNA primers. For assessment of plasmid-mediated quinolone resistance, isolates were screened for qnrA by PCR using specific primers. PCR was performed for all 100 clinical isolates on a Bio-Rad T100 thermal cycler. Amplified products were visualized following electrophoresis on 2% agarose gel, and each isolate was categorized according to the presence or absence of the expected amplicon. The study had ethics approval from the Ethics Committee of Hamadan University of Medical Sciences (IR.UMSHA.REC.1402.090).

Results

Patients ranged in age from 20 to 94 years, with a mean age of 54 years. The most frequent age category was 60-74 years, comprising 40% of participants. Women constituted the majority of the sample: 73 urine isolates were obtained from women and 27 from men. All 100 clinical isolates were confirmed as E. coli by phenotypic and molecular methods. Antimicrobial susceptibility testing demonstrated substantial resistance to each of the three quinolones evaluated. Nalidixic acid showed the highest resistance, with 70 isolates (70%) classified as resistant, 7 (7%) as intermediate, and 23 (23%) as susceptible. Ciprofloxacin resistance was detected in 59 isolates (59%), with 4 isolates (4%) intermediate and 37 (37%) susceptible. Levofloxacin resistance was observed in 53 isolates (53%); no isolate was intermediate, and 47 isolates (47%) were susceptible. The original Table 2, translated into English and retaining its source numbering and numerical values, summarizes susceptibility categories by sex and total frequency.

Table 2. Frequency and percentage of susceptible, intermediate, and resistant Escherichia coli isolates from urinary tract infection samples to the evaluated antibiotics.

More than 40% of the clinical isolates were resistant to all three tested agents. Among the evaluated antibiotics, the highest susceptibility was observed for levofloxacin (47%), followed by ciprofloxacin (37%) and nalidixic acid (23%). PCR screening showed that the qnrA gene was present in 28% of the 100 clinical E. coli isolates. The article reports that qnrA-positive isolates were found among isolates resistant to the studied drugs, supporting a possible contribution of this gene to quinolone resistance. In comparison with earlier studies cited by the authors, ciprofloxacin resistance in the present series (59%) exceeded the 33% reported in Hamadan by Hashemi et al. [20] and the 20.8% reported by Sedighi et al. [21], whereas another Hamadan study reported higher resistance values for nalidixic acid, ciprofloxacin, and ofloxacin [22]. The observed qnrA frequency of 28% was also higher than values reported in Egypt (6.7%) [23] and Mexico (22.7%) [24], absent in the earlier Hamadan study by Sedighi et al. [21], and close to the 29% reported by Karshenas et al. in isolates from Hamadan hospitals [25]. The source article attributes variability among studies to differences in sampling time and location, patterns of antibiotic use, and the frequency of genetic transfer mechanisms.

Conclusion

The study identified a high level of quinolone resistance among urinary E. coli isolates from hospitalized patients in Hamadan: 70% were resistant to nalidixic acid, 59% to ciprofloxacin, and 53% to levofloxacin, while qnrA was detected in 28% of isolates. These findings support the authors' conclusion that qnrA may be one factor contributing to quinolone resistance. In view of the high resistance documented in this series and the article's discussion of recent local findings, empirical use of quinolones alone for uncomplicated UTI was not recommended in the studied hospital context; the authors emphasized rational antibiotic prescribing, close treatment follow-up with urine culture, and consideration of combined therapy where clinically appropriate.

Keywords: Antibiotic Resistance, Escherichia coli, Quinolones, Urinary Tract Infection

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