Antimicrobial Susceptibility and Molecular Detection of Integrons, Sulfonamides and Trimethoprim Resistance of Extra Drug-Resistant Escherichia coli Isolates

Background: Escherichia coli, a gram-negative bacterium, is the causative agent for approximately 80% of urinary tract infections (UTIs). UTI treatment has resulted in the overuse of antibiotics in hospitals and communities, and subsequently the increase of antimicrobial resistance. The emergence of extensively drug resistance (XDR) strains has become a costly and dangerous challenge in the treatment of most bacterial infections and UTIs. Objective: This study aimed to determine the frequency of XDR isolates and investigate the distribution of common sulfonamide-(sul1, sul2, & sul3) and trimethoprim (dfrA1, dfrA12, & dfrA14) -related resistance genes among E. coli isolates from UTI patients. Furthermore, the isolates were sought for the presence of class 1 and class 2 integrons (Int1 & Int2) among XDR E. coli isolates. Materials and Methods: 120 uropathogenic-E. coli isolates recovered from UTI cases in Mashhad were assessed in 2017-2019. Overall, 39 out of 120 isolates were identified as XDR isolates as they were resistant to all classes of tested antibiotics, except for two or fewer comprising quinolones (first and second generation), cephalosporins (first and third generation), penicillins, tetracyclines, and sulfonamide-trimethoprim. Results: The antimicrobial susceptibility testing (AST) results determined a substantial resistance rate against cloxacillin (98.3%), oxacillin (98.3%), and cephalexin (94.17%). According to polymerase-chain reaction results, sul1 and dfrA14 genes with the frequency of 35 (89.74%) and 28 (71.79%) were identified as the most prevalent resistant genes among XDR isolates. In addition, int1 and int2 genes were detected among 23 (58.9%) and 8 (20.5%) XDR isolates, respectively. In conclusion, the substantial distribution of sul1 and dfrA14 genes was highlighted among XDR E. coli isolates recovered from UTI. Conclusion: Based on the present research findings, class I integrons play a major role in the dissemination of resistance gene cassettes, including sul and dfr in XDR isolates, and should be investigated in the future.

consists of nitrofurantoin monohydrate/macrocrystals and trimethoprim-sulfamethoxazole. 10 Patients should be treated with first-line antibiotics that among the local Escherichia coli population, have a good effect on preventing further development of MDR, XDR, and PDR.However, to make the best possible empirical choice, it is crucial to periodically assess antibiotic susceptibility in commonly treated populations with UTIs. 2,11n the other hand, the mainly acquired resistance mechanisms against sulfonamide and trimethoprim have been defined through mutations and alterations in genes encoding the dihydropteroate synthase or dihydrofolate reductase as target enzymes or by harboring the responsible genes for encoding dihydropteroate synthetases (sul) or insensitive dihydrofolate reductases (dfr) that are insensitive to sulfonamide and trimethoprim, respectively. 12Further, integrons (Int) are highly reported as important factors that carry resistance gene cassettes that increase the dissemination of antimicrobial resistance genes. 13Thus, multiple sul and/or dfr genes on integrons contribute to spreading resistance. 12owadays, physicians in Iran rarely prescribe firstline treatment alternatives, which may worsen resistance development and limit our choice to save new drugs for the last resort treatment in complicated cases.As a result, it appears necessary to evaluate whether first-line alternatives such as sulfonamides and trimethoprim are still effective against uropathogenic E. coli.Therefore, the current study sought to determine the frequency of XDR isolates and the rate of common sulfonamide-(sul1, sul2, & sul3) and trimethoprim (dfra1, dfra12, & dfra14)-related resistance genes in E. coli isolates from UTI patients in Mashhad, Iran in 2017-2019.In addition, class 1 and class 2 integrons (Int1 & Int2) were looked for in XDR E. coli isolates.

Bacterial Sample Collection
The current study was conducted on 120 uropathogenic-E.coli (UPEC) isolates recovered from uncomplicated UTI cases.All isolates were collected from patients with clinical UTIs in 2017-2019.The etiologic pathogen was then identified as E. coli using the biochemical standard tests and detecting a specific gene, namely, cdgR (a cyclic di-GMP regulator) gene for E. coli according to previous research 14 .Eventually, the identified E. coli isolates were stored at a -70℃ refrigerator in trypticase soy broth, and 15% glycerol was added to them.

Antimicrobial Susceptibility Testing
The antimicrobial susceptibility of isolates was evaluated against nine antibiotics using the Kirby-Bauer disk diffusion method as recommended by Clinical & Laboratory Standards Institute (CLSI) 2021. 15The nine tested antibiotics were cefalexin (30 μg), ceftriaxone (30 μg), cefotaxime (30 μg), nitrofurantoin (300 μg), nalidixic acid (30 μg), ciprofloxacin (5 μg), norfloxacin (10 μg), doxycycline (30 μg), and trimethoprimsulphamethoxazole (1.25/23.75μg).E. coli ATCC 25922 was implied as a positive control strain.The interpretation of antimicrobial susceptibility testing (AST) was accomplished according to the CLSI 2021.The XDR isolates were determined regarding non-susceptibility to at least one agent in all but two or fewer antimicrobial categories (i.e., bacterial isolates remained susceptible to only one or two categories) for the investigation of the frequency of resistance genes. 16

Molecular Detection of Antimicrobial Resistance
First, the DNA extraction of sulfonamide resistance isolates was performed using the boiling method based on an earlier study. 17The multiplex-polymerase chain reaction (m-PCR) was set up for the molecular determination of sulfonamide resistance genes (sul1 & sul2); moreover, another sulfonamide resistance gene (sul3), the integrons (Int1, 2), and trimethoprim resistance genes (dfrA1, dfrA12, & dfrA14) were detected through uniplex-PCR (u-PCR).The PCR was set up at the final 25 μL, consisting of 12.5 μL of PCR 2 × Master Mix (Amplicon, Denmark) containing Taq DNA Polymerase, reaction buffer (including Tris-HCL, potassium chloride, and magnesium chloride), and dNTPs mixture, a protein stabilizer, and the convenience for use was optimized by adding sediment for electrophoresis and 2 × solution of loading dye, 0.5 μL of each primer (2 µM), 2 μL of template DNA, and up to 25 μL the final volume used nuclease-free water.Further PCR information, including the oligonucleotide primer sequences and annealing temperature, is listed in Table 1.

Statistical Analysis
Analyses were accomplished using SPSSTM software, version 22.0 (IBM Corp., USA).The results are presented as descriptive statistics in terms of the relative frequency.Values were expressed as the mean ± standard deviation or group percentages (categorical variables).Fisher's exact statistical test was performed to analyze the data, and P ≤ 0.05 was considered statistically significant.

Molecular Confirmation of Escherichia coli Strains
All 120 isolates were confirmed by amplifying the cdgR (cyclic di-GMP regulator) gene using the PCR method.

Discussion
Over 150 million cases of UTI diseases per year have been reported worldwide, subsequently causing a 6-billion-US dollar cost for treatment. 22,23UTIs have been managed using oral antibiotics consisting of cephalosporins, trimethoprim-sulfamethoxazole, and fluoroquinolones. 24owever, overusing these antibiotics has recently developed resistant strains against mentioned antibiotics that restricted antibiotic therapy. 25Indeed, the appropriate treatment of UTIs is one of the most serious problems regarding raising MDR, XDR, and PDR strains.
In the current study, 39 out of 120 (32.5%)6][27] However, Yuan et al reported a high frequency of XDR (64%) in China. 28Interestingly, in a comprehensive Australian laboratory-based retrospective assessment, Fasugba et al demonstrated the 0.2% XDR rate among UTI E. coli isolates during a 5-year assessment. 29This comparison has shown that observation and appropriate antibiotic supervision can reduce and control increased antimicrobial resistance.
Considering the AST results, a significantly resistant rate of UTI isolates was revealed against β-lactam families (oxacillin, cephalexin, and cloxacillin, > 94%).On the other hand, the highest activity was observed for co-trimoxazole, ciprofloxacin, and norfloxacin with a sensitivity of 43.3%, 42.5%, and 41.7%, respectively.These findings are correlated with those of a recent meta-analysis study by Jabalameli et al, representing the high distribution of extended-spectrum β-lactamase producing E. coli in Iran. 30However, the rate of resistance against β-lactams families was reported as the lowest one in another meta-analysis survey by Bunduki et al, analyzing the AST reports of UPEC worldwide. 13Several reasons such as differences in tested antibiotics and sample   sizes can explain this controversy.Regarding the present findings, co-trimoxazole, ciprofloxacin, and norfloxacin might be practical choices for empirical therapy.
The PCR results confirmed a co-harboring of sul1 and sul2 genes among 15 (38.4%)XDR isolates.In contradiction with earlier studies that reported 44.150.6%,respectively, 31,32 our finding is in line with that of another study in southwest Iran by Boroumand et al, reporting the frequency of co-existing sul1 and sul2 genes by 37.3%. 33urprisingly, the dfrA14 gene was the predominant gene (71.7%)among different tested variants of dfrA genes.As reported in prior surveys, the dfrA14 gene was common within isolates from animals 20,34 ; consequently, the dfrA14 gene is disseminated among E. coli strains in the environment, which raises resistance strains.
As a classical structure, integrons are involved in the bacteria evolution through reading cassette frameworks. 35n the current survey, 26 out of 39 XDR isolates (66.6%) contained either Int1 or/and Int2; this supported the statement that integrons exert a highlighted role in developing antimicrobial-resistant, particularly among Gram-negative bacteria.36 No significant difference was found with the earlier experiment that reported 76.7% positive isolates for the presence of integrons 37 ; Further, the high prevalence of intl1 was observed among isolates in several studies.[38][39][40] However, the present study reported a lower incidence of intl1 compared with the mentioned studies.Nevertheless, as previously reported by Khamesipour and Tajbakhsh, class 1 integrons with different gene cassette arrays and sul1 genes were highly common in Enterobacteriaceae; likewise, the remarkable co-existence of sul1 or sul2 genes among Int1 positive isolates has been determined in a currently running survey.41 This observation has been confirmed with earlier studies.32,42 Although all Int2 positive isolates were dfra14 gene positive as well, there was no significant relationship between the presence of these genes.However, a literature review study by Sabbagh et al introduced the dfra14 gene as a new cassette gene for Int2.35 The most important reason for these differences in the obtained results was the difference in the number of tested strains.In most studies, more than 100 strains have been used, while in the present study, the number of strains was 39.
Further experiments are essential for analyzing and characterizing integrons' structures and determining the relationship of cassette genes with integrons.

Conclusion
The results have outlined the most activity for cotrimoxazole and quinolones among tested antibiotics, indicating that they can still be helpful as first-line treatment.However, a significant rate of XDR phenotype among E. coli isolates from UTI patients was detected as well.The highlighted prevalence was demonstrated for sul1 and dfrA14 genes among XDR isolates.Regarding the high distribution of integrons within tested isolates, it can be concluded that integrons influence the dissemination of the dfrA14 gene among E. coli isolates from different sources.

Table 1 .
The Sequence of Oligonucleotides Used as Primers

Table 2 .
The Antimicrobial Resistance Patterns of XDR Escherichia coli Isolates From UTI Patients (n = 39) Note.XDR: Extra drug resistance; E. coli: Escherichia coli; UTI: Urinary tract infection.

Table 3 .
Prevalence of sul, dfr, Int class 1 and Int Class 2 and the Association of Class 1 and 2 Integrons With sul 1, 2 and dfrA 1, 12, 14 Resistance Genes Among XDR Escherichia coli *