Evaluation of adhesin genes and risk factors associated with urinary tract infections by drug-resistant uropathogenic Escherichia coli in North of Iran

Background: Uropathogenic Escherichia coli (UPEC) isolates, have a wide variety of virulence factors to promote colonization and survival in the urinary tract. This study aimed to evaluate adhesin genes, biofilm formation ability, antibiotic resistance profiles of UPEC strains, and the related risk factors in patients with UTIs caused by drug-resistant UPEC. Methodology: A total of 105 UPEC isolates were evaluated for biofilm formation using 96-well microtiter plates, the presence of adhesin genes by PCR assay and the antimicrobial susceptibility pattern using the disk diffusion method. Demographic and clinical characteristics of patients were investigated to identify predisposing factors for drug-resistant isolates. Results: Out of 105 UPEC isolates, 84.8% were positive for biofilm formation. Biofilm-producing isolates exhibited a significantly higher prevalence of fimH , kpsMTII , csgA , afa/draBC , and pap adhesin genes compared to non-biofilm-producing strains ( p < 0.05). The results also revealed that 52.4% of the isolates were ESBL-producing, and 84.8% were multidrug-resistant (MDR). Further analysis of antibiotic susceptibility among ESBL-producing strains showed the highest resistance rates to ampicillin, ciprofloxacin, and trimethoprim-sulfamethoxazole. Conversely, the highest susceptibility, in addition to carbapenems, was observed for fosfomycin, amikacin, cefoxitin, and nitrofurantoin. We identified hypertension as a potential risk factor for infection with ESBL-producing UPEC strains. Conclusions: Our results revealed a significant rate of drug resistance among UPEC isolates obtained from UTIs in our region. This underscores the importance of monitoring the empirical use of antibiotics and identifying specific risk factors in our geographical area to guide the selection of appropriate empirical treatment for UTIs.


Introduction
Urinary tract infections (UTIs), as a global health problem, are one of the most common bacterial infections, affecting a wide range of hospitalized and outpatients with high mortality and morbidity rates worldwide [1][2][3].Uropathogenic Escherichia coli (UPEC) is the major cause of UTIs in 70-95% of community-acquired cases and 50% of all nosocomial infections [4,5].UPEC encodes various virulence factors, such as toxins, capsules, invasins, and adhesins, which contribute to attaching to uroepithelial cells, and development and severity of UTIs [6,7].Surface virulence factors (adhesins) such as P fimbriae (pap), Type1 fimbriae (fim), afimbrial adhesins (afa), and polysaccharide surface structures (capsule) are the main attachment factors increase the ability of bacteria to colonize and formation of biofilm and consequently persist in the urinary tract, which is associated with recurrence of urinary tract infections [2,8,9].In addition, pga genes are required to synthesize related polysaccharide adhesins and optimal biofilm formation under various growth conditions [10,11].Csg operons as regulators affect the expression of the curli genes [12,13].Antigen 43 (flu), as a prominent surface protein in E. coli, with auto-aggregation and microcolony formation through cell-to-cell interactions, promotes biofilm formation [14,15].Also, biofilm, as a sticky exopolysaccharide matrix, can cause of restricting the binding of antimicrobials and the exchange of plasmids between bacteria, which develops resistance against a broad spectrum of antibiotics [11,16,17].Moreover, excessive and inappropriate use of antibiotics contributes significantly to the increasing rate of multiple-drug resistant (MDR) strains commonly related to increased extended-spectrum beta-lactamases (ESBLs)-producing bacterial strains.The prevalence of ESBLs-producing UPECs is raising in urinary tract infections [11].In addition, ESBLs-producing UPEC harbors resistance to a broad spectrum of -lactam antibiotics, cephalosporins, monobactams, aminoglycosides, quinolones, tetracyclines, nitrofurantoin and trimethoprim-sulfamethoxazole [6].
Currently, the emergence of MDR and ESBLsproducing strains among UPEC isolates has increased as a major problem in treating urinary tract infections, leading to limited therapeutic choices and a serious threat to global health.Therefore, identifying risk factors for antimicrobial resistance may improve the empirical treatment of UTIs caused by ESBLsproducing and MDR UPEC strains [18].
The multidrug resistance observed among UPEC strains significantly restricts therapeutic options and poses a life-threatening risk, with a profound impact on human health worldwide.The aim of the present study was to assess colonization-associated genes, biofilm formation ability, antibiotic resistance profiles of ESBLs-producing UPEC strains isolated from patients with UTIs, along with their associated risk factors.The findings of this study will offer vital epidemiological insights into UPEC strains, which will be instrumental in devising more effective strategies for the prevention, control, and treatment of UTIs in the investigated region.

Study design and bacterial identification
This cross-sectional study was conducted over a ten-month period from January to October 2021 in the department of Microbiology, Virology and Microbial Toxins, faculty of medicine at Guilan University of Medical Sciences, Rasht, Iran.This study was approved by the Research Ethics Committee (IR.GUMS.REC.1399.603)at Guilan University of Medical Sciences in Rasht, Iran.UPEC isolates were obtained from urine samples collected from patients with UTIs who were referred to Razi Teaching Hospital.Then, the isolates were identified using standard bacteriological and biochemical tests.The pure colonies were stored in trypticase soy broth supplemented with 20% glycerol at -80°C for later use.

Phenotypic detection of biofilm production in UPEC
The biofilm formation assay of UPEC isolates was carried out using 96-well microtiter plates according to Stepanovic et al. with some modifications.Briefly, isolates were grown overnight in trypticase soy broth (TSB) at 37°C.The cultures were adjusted to 0.5 McFarland standard turbidity and diluted 1: 100 with Mueller-Hinton Broth (MHB).Then 200 µL of diluted suspension was added to each well of the microplate (Maxwell, China), supplemented with 2% glucose (Merck, Germany), and incubated at 37°C for 24 hours.MHB containing 2% glucose was considered as a negative control.After the incubation, the content of the wells was discarded and washed with distilled water three times to remove non-adherent bacteria.The adhered cells were stained with 125 μL of 0.1% (w/v) crystal violet for 15 minutes.Then, the dye was discarded, and the microplate was washed three times and dried at room temperature.Furthermore, 125 μL of 33% glacial acetic acid (Merck, Germany) was added to the wells to release the dye.Subsequently, samples were transferred to another microplate, and the absorbance was measured at 570 nm using an ELISA reader.UPEC strains were categorized according to the criteria of biofilm formation ability introduced by Stepanovic et al. as a non-biofilm producer, weak biofilm producer, moderate biofilm producer, or strong biofilm producer [21].All assays were performed in triplicate, and results are given as the mean of at least two independent experiments ± SD.

DNA extraction and detection of adhesin genes by PCR assay
Chromosomal DNA of all isolates of UPEC was extracted by boiling method.Briefly, 500µL aliquots of overnight Brain-Heart Infusion broth (Merck, Germany) cultures were centrifuged at 14,000 rpm for 10 minutes at room temperature.The cell pellets were resuspended in 400µL of 1 × Tris-EDTA buffer, heated at 95 °C for 10 minutes, and placed at room temperature for 5 minutes.Samples were placed at -20 °C for 10 minutes, and after centrifugation at 14,000 rpm for 10 minutes at 4 °C, the supernatant was used as the DNA template.The concentration of DNA was measured using Nano drop.Amplification of different adhesin genes was performed by conventional PCR using specific primers (TAG Copenhagen A/S, Denmark).Primer sequences, predicted sizes of the PCR products, and amplification conditions are shown in Table 1.PCR reactions were done in a final volume of 20 μL containing 10 μL master mix (2 × PCRBIO Taq Mix Red -PCR BIOSYSTEMS), 1 μL of each primer (10 pmol/μL), 1 μL of extracted DNA and sterile Double distilled water to reach to 20 μL.The PCR conditions consisted of an initial denaturation step at 95 °C for 5 mins, followed by 35 cycles of DNA denaturation at 95 °C for 30 seconds and primer annealing for 30 seconds.Temperature depended on the sequences of primers, and primer extension at 72°C for 1 minute, followed by a final extension at 72 °C for 7 minutes.Amplicons were analyzed by electrophoresis on a 1.5% agarose gel.

Data analysis
Statistical analysis was performed using SPSS™ software, version 21.0 (IBM Corp., Armonk, NY, USA).The results are presented as descriptive statistics in percentage base distribution.Statistical analysis was carried out using Chi-square (χ 2 ) test to evaluate the relationship between the variables.A p value < 0.05 was considered statistically significant.
Antibiotic resistance of the UPEC isolates was investigated using ten antibiotics classes.Out of 105 UPEC strains, 55 (52.4%) were ESBLs-producing.The resistance pattern was compared between ESBLs and non-ESBLs-producing strains and are shown in Table 2.The ESBLs-producing strains showed significantly (p < 0.05) a higher degree of resistance to ampicillin, cefazolin, cefuroxime, cefepime, ceftriaxone, piperacillin-tazobactam, gentamicin, ciprofloxacin, trimethoprim-sulfamethoxazole, levofloxacin and azithromycin as compared to non-ESBLs-producing strains.Among the ESBLs-producing isolates, the highest resistance rate was observed for cefuroxime (100%), followed by ampicillin (96.4%) and ciprofloxacin (94.5%).However, the highest susceptibility was toward imipenem and meropenem (96.4%), amikacin (87.3%), and cefoxitin (83.6%).Also, the frequency of MDR isolates was recorded 84.8%.The rate of MDR isolates was significantly Biofilm production assay showed that 89 (84.8%) and 16 (15.2%) of UPEC isolates were biofilm producers and non-biofilm producers, respectively (Table 3).In this study, we considered strong and moderate biofilm producers as biofilm producer, weak and no biofilm producers as no biofilm producer.In the present study, there was a prevalence of 53.9% and 85.4% ESBL-producing and MDR in biofilmproducing isolates, respectively.Among the 83 patients with recurrent UTI, 70 (84.3%)UPEC isolates were biofilm producers and of the 22 patients with norecurrent UTI, 19 (86.4%)UPEC isolates were biofilm producers.According to our results, there was no  significant correlation between biofilm production and recurrent UTI.
The results of the prevalence of adhesin genes among biofilm producers and non-biofilm producers of UPEC isolates are shown in Table 3.Among adhesins, the most prevalent genes among all the biofilm producer isolates were fimH (100%) followed by kpsMTII (78.7%) and csgA (68.5%).Also, the frequency of 4 adhesin genes (papC, papGII, afa/draBC and pgaC) was more than 50% among the biofilm producer isolates.
In our study, analysis of the risk factors for ESBLsproducing isolates showed that hypertension could predispose patients to infection by an ESBLs-producing UPEC strains (Table 4).

Discussion
The increasing resistance to the broad spectrum of antibiotics among ESBLs-producing and MDR UPEC strains is a global challenge for appropriate treatment of UTIs [18,22].As an essential virulence factor, biofilm formation enhances antibiotic resistance and results in bacterial persistence and chronic infections [7,[23][24][25].According to the recommendation of the America Infectious Diseases Society, monitoring the empirical use of antibiotics at a community level or geographic region is vital to select a proper treatment for UTIs [26].Therefore, we report biofilm formation's ability, the antibiotic resistance pattern of ESBLs-producing and MDR UPEC isolates, and the related risk factors for UTIs in the north of Iran.
Consistent with the previous studies in Iran and other countries, in our results, 52.4 and 84.8% of UPEC isolates belonged to ESBLs-producing and MDR strains, respectively [27][28][29][30][31].The prevalence of MDR isolates was higher than the pooled prevalence of MDR UPEC with 49.4% [32] and 65.8% [33] in Iran.Also, according to previous reports, the relative frequency of ESBLs-producing isolates varied from 24% to 72.9% in different regions of Iran [34].Our findings showed a high antibiotic resistance rate among both ESBLsproducing and MDR isolates compared to non ESBLsproducing and non-MDR isolates.In agreement with some results from different parts, most ESBLsproducing and MDR isolates were resistant to ampicillin, ciprofloxacin and trimethoprimsulfamethoxazole, which preclude their use as the firstline empirical therapy of UTIs [31,35,36].
Increased antibiotic resistance during recent years in different geographical areas of Iran has created challenges in the empirical treatment of UTIs due to differences in infection control policy and inappropriate and overuse of antibiotics [37].
On the other hand, our results revealed that not only were the majority of ESBLs-producing and MDR isolates susceptible to imipenem, meropenem, and fosfomycin but susceptibility to amikacin, cefoxitin, and nitrofurantoin was also considerable.This finding is consistent with some previous reports from Iran and other countries [29,31].As carbapenems are β-lactam antibiotics with high antibacterial activity, they are commonly used as first-line treatments for infections caused by ESBLs-producing Enterobacterales.However, to prevent the emergence of carbapenemresistant strains, it is essential to consider the use of alternative drugs [38,39].Hence, to preserve carbapenems as valuable antibiotics, and based on our findings, oral options for treating UTIs caused by drugresistant UPEC strains in our study region include nitrofurantoin and fosfomycin [36].
Most of our UPEC isolates showed the ability of biofilm formation (84.8%), in similar to other published reports [7,30].In agreement with previous studies, our results according to Table 3, showed no significant correlation between biofilm formation and ESBLs production and MDR [11,30].On the contrary, some reports found a positive correlation between biofilm formation and drug resistant [40,41].These results reveal that although antibiotic resistance mechanisms may be associated with the formation of biofilm among UPEC strains, the development of antibiotic resistance in UPEC strains may be due to natural selection pressure caused by excessive and inappropriate use of antibiotics, exchange of plasmid-mediated genes, etc. [42][43][44].
Pathogenic UPEC strains encode a variety of adhesin genes, including fimbrial adhesins (such as Type 1 and P), afimbrial adhesins (afa), and polysaccharide surface structures (kps), which are among the most common virulence factors.These genes enable the bacteria to colonize and survive in the urinary tract [8,9].
In agreement with some previous studies, our results demonstrated a significant association (p < 0.05) between biofilm production and the presence of fimH, kpsMTII, pap genes, afa/draBC, and csgA adhesin genes [17,44,45].All 54 papGII-positive isolates, were biofilm producers.These results show a possible relationship between the presence of this gene and biofilm formation in UPEC isolates.Nonetheless, the results of some studies similar to this study showed no significant correlation between biofilm production and frequency of flu and pgaC genes [11,24,46].It may be due to the involvement of various adhesin genes and their expression in different types of UPEC strains, i.e., csg, fimH, afa, pap locus, kps, etc., responsible for the production of biofilm [47,48].In addition, a mutation in the target gene could be an important reason for the lack of correlation between the presence of these genes and biofilm formation [49].
In our study, most of the patients were categorized as community-acquired urinary tract infections (CA-UTIs) and we identified that hypertension might be a risk factor for infection with ESBLs-producing UPEC strains.Previous studies have evaluated risk factors for UTIs caused by multidrug-resistant UPEC strains.A large study from Scotland involving 40,984 isolates demonstrated that increasing age is the main risk factor for multidrug resistance (MDR) due to the weakening of the immune system with aging, making the treatment of UTIs more difficult and recurrence more frequent [50].Other studies have also shown male gender [51][52][53], older age [22,26,52,53], urinary catheterization [18,26], history of antibiotic use in the last three months [22,52], previous hospitalization [22,[54][55][56], diabetes mellitus [54][55][56][57], and recurrent UTI [52] as predisposing factors that increase the risk of urinary tract infections by multidrug-resistant bacteria.
There are several limitations to the present study.First, this is a study of 105 UPEC strains over a 10month period in a single hospital, limiting the scope of the study.Second, survey to the papG alleles and all of the effective genes in adhesion and the expression of them.Also, to evaluate risk factors, similar groups of patients in a large population size could be considered.

Conclusions
This study highlights the analysis of antimicrobial resistance, showing a high level of resistance to the first-line empiric treatments for UTIs caused by UPEC isolates in our studied region.Our finding revealed that, in addition to carbapenems, most ESBLs-producing strains were sensitive to fosfomycin, amikacin, cefoxitin and nitrofurantoin.Although the majority of isolates were biofilm producers, no significant association was found between biofilm formation and drug resistance.The presence of fimH, pap genes, kpsMTII, afa/draBC, and csgA adhesin genes appears to be associated with a high potential for biofilm formation.We identified that hypertension might be a risk factor for infection with ESBLs-producing strains in our studied region.Finally, our findings demonstrate the importance of monitoring empirical use of antibiotics and noticing certain risk factors in our geographical area to select an appropriate empirical treatment for UTIs.

Table 1 .
List of primers used in this study.

Table 3 .
Correlation between adhesin genes among biofilm producer and non-biofilm producers of UPEC isolates.

Table 4 .
Risk factors and demographic characteristics of patients with infection with ESBL-producing UPEC strains.