Iranian Journal of Medical Microbiology

Collection, Isolation, and Identification

In this descriptive-analytical study, during 3 months (from February to May 2017), 100 urine samples from pregnant women suspected of UTI were collected from comprehensive health centers in Karaj, Iran. To isolate E. coli and ensure the purity of the samples, each sample was cultured on McConkey agar and nutrient agar medium, and the presence of E. coli was confirmed by routine microscopic and biochemical tests.

Determination of Antibiotic Susceptibility by Disk Diffusion Method

Kirby-Bauer disk diffusion method was performed to determine the antibiotic susceptibility of E. coli isolates against six different antibiotic classes in Mueller-Hinton agar (Merck, Germany) according to the CLSI recommendations (2017). The antibiotic disks included ampicillin (AM, 10μg), cotrimoxazole (SXT, 25μg), ciprofloxacin (CP, 5μg), gentamicin (GM, 10μg), nitrofurantoin (FM, 300μg), and amikacin (AN, 30μg) (Padtan Teb Co, Iran). Reference strains of E. coli ATCC 25922 were used as quality control of the antibiotic disks (10).

Evaluation of Biofilm Formation in UPEC by Microtiter Plate Method

In this method, a 24-hour culture of bacteria in Luria Bertani (LB) liquid medium was dilute as 0.5 McFarland turbidity and pour 10 μL into 990 μL of sterile LB medium to prepare a 1% dilution and then 200 µL was added to three cavities of the plate. After 24 hours of incubation at 37°C, first, it was washed with saline phosphate buffer three times at pH 7.3 to remove the bacteria that are not connected to the plate wall. It is then fixed with 250 μL of pure methanol for 15 minutes. Then, 200 µL crystal violet staining 2% was added and after 5 minutes, it was washed with phosphate-buffered saline. After that, 160 µL of glacial acetic acid 33% v / v was added to every well, after 15 minutes of incubation of the plates at 37°C, the light absorption of stained wells was detected with a wavelength of 570 nm in the ELISA reader (Stat Fax - 4200).
 The results were divided according to Table 1, without the ability to form biofilms (-), weak (+), medium (++), and high strength (+++). To increase the accuracy of the experiment, each sample was repeated three times and the average obtained was considered as the final result of the experiment (4).

DNA Extraction for Duplex PCR

The boiling method was used to extract the DNA content of bacterial isolates (4). After extracting the DNA, evaluated qualitatively and quantitatively using a spectrophotometer, and electrophoresis was carried out on 1% agarose gel. The extracted DNA was stored at -70°C until further analysis.

Duplex PCR Reaction for the Detection of papC and sfa Genes

Amplification reactions were carried out in 25 µL volumes comprising; one microliter of target DNA (10 ng / μL ), 12.5 μL of dye Master mix2X (CinaGen, Co., Tehran, Iran), 1 μL of each Forward and Revers primers (20 p/mol) (Forward primers for papC F 5́ -GACGGCTGTACTGCAGGGTGTGGCG-3 and Revers PapC R 5́- ATATCCTTTCTGCAGGGATGCAATA-3 and Forward primers for sfa F 5́- CTCCGGAGAACTGGGTGCATCTTAC-3 and Revers primers sfa R 5́- CGGAGGAGTAATTACAAACCTGGCA-3) and 9.5 μL double distilled water.
Each program includes 30 cycles of PCR amplification under the following conditions: Initial denaturation at 94°C for 3 minutes, 30 thermal cycles including denaturation at 94°C for 1 minute, annealing at 59°C for 45 seconds, extension at 72°C for 1 minute and final extension it was 72°C for 5 minutes. The amplified products were visualized after electrophoresis on a 1% agarose gel and finally evaluated with a UV transilluminator. In the PCR test, distilled water was used as a negative control and the reference strain of E. coli ATCC 10536 was used as a positive control.

Statistical Analysis

The results were evaluated by Excel 2010 (Microsoft Office, Microsoft, Washington D.C, USA) and SPSS 2016 (SPSS Inc., Chicago, IL, USA) and the non-independence test in inferential statistics to investigate the relationship between qualitative variables. P-value≤0.05 was analyzed as the significance level.

Figure 1. The results of electrophoresis of PCR products to identify genes papC (328 bp) and sfa (410 bp), wells 1, 2, 4, 5, 6, and 8 are positive for both genes papC (328 bp) and sfa (410 bp), and wells 3 and 7 are positive only for papC (328 bp).

Uropathogenic E. coli pathotypes are responsible for 70-90% of community-acquired UTI and 50% of hospital-acquired UTI (12). UPEC damages the host tissue by colonization and biofilm formation in the mucosal epithelium. The attachment of urogenital bacteria to the epithelial cells is usually very important for biofilm formation because these bacteria do not wash out as quickly as other bacteria (13). This bacterium can form intercellular aggregates similar to biofilm structures within the bladder epithelium; therefore, biofilm formation plays an important role in the pathogenesis of UPEC (9). The papC and sfa genes are the most common genes encoding pili in E. coli isolated from UTI that can help bind the bacterium to host tissues and form antibiotic-resistant biofilms. Identification of biofilm-producing UPEC strains is important to better understand the pathogenicity and antibiotic resistance of this bacterium in UTI. On the other hand, determining the antibiotic resistance pattern of E. coli causing UTI to identify effective drugs in the initial treatment of disease and the emergence of resistance can be important in controlling urinary tract infections. In different parts of Iran, several studies have been performed on E. coli strains isolated from UTI (9); however, the overall results on the significant and pervasive resistances or emerging resistances of E. coli are not available in the country. Also, the trend of changing the pattern of antibiotic susceptibility of this main pathogen of the urinary tract in the country is not known. Considering the aforementioned points, the study was done on 100 samples of urine collected from pregnant women suspected of UTI, with ages between 20 and 25 years. Based on the results, E. coli was isolated from 64 (64%) samples obtained from patients suspected of UTI. These results show that E. coli is still the most common cause of urinary tract infections. In this study, the resistance of UPEC bacteria causing UTI to antibiotics was investigated. Based on the results; the highest levels of resistance were related to cotrimoxazole (40.6%), ampicillin (31.3%), ciprofloxacin (12.5%), gentamicin (7.8%), nitrofurantoin (4.6%), and amikacin (3.1%), respectively, which is consistent with the results of a study conducted by Mattai et al. in 2004 (14). E. coli isolated in this study showed the highest sensitivity to amikacin (96.9%), which is consistent with the study of Abdollahi Kheirabadi et al., in 2012, who reported the sensitivity of E. coli isolated from urinary specimens to amikacin (98%) (15). Also, in a study conducted by Milani et al., on the antibiotic susceptibility of bacteria isolated from people with UTI, the highest resistance was to ampicillin (95.3%) and the lowest resistance was to amikacin (6.6%) (16). According to the results of this study, the level of resistance to ampicillin was higher than our findings, but in our study, most of the isolates were sensitive to amikacin. The results of the present study showed that more than 47% of the isolates were resistant to more than two groups of antibiotics. In a study conducted by Eslami et al. in Iran in 1995, 85.5% of isolates showed resistance to more than two antibiotics (17). In a study by Molina_Lopez et al., (2011) in Mexico City, multidrug resistance strains of UPEC showed similar results (18). In their study, the lowest resistance was to amikacin and nitrofurantoin, which is consistent with the results of the present study (18). In the study of Mansouri et al., the resistance to ciprofloxacin was 41%, gentamicin was 34.8% and cotrimoxazole was 93.5%, which has a higher resistance compared to the present study (19); this may be due to differences in geographical areas and sources from which the bacterium has been isolated. One of the factors that play a role in pathogenesis as well as resistance to antimicrobial agents is the potential of biofilm formation. The first step in the process of colonization or infection is the attachment of bacteria to the host cell. In this study, the strength of biofilm formation was investigated by the microtiter plate method, which is a standard qualitative method (4).
In this study, 48.4% of the isolates were strong biofilm producers, 15.6% were moderately potent, 21.8% were weak and 14.2% were not biofilm producers. In the study of Sevanan et al. in 2011, they used the biofilm formation method in the tube (Tube Method) and showed that 9.4% of the isolates were high strength biofilm producer, 34.4% of the isolates were moderately potent, and 40.6% of the isolates with weak power could produce biofilm and 15.6% could not produce biofilm; These results showed more biofilm formation compared to microtiter plate method (20). Rawa’a Al-Chalabi et al. also reported in 2010 that 90% of UPEC strains can form biofilms (21). In a 2012 study by Ponnusamy et al., out of 100 strains of UPEC, 72 strains showed positive biofilm phenotypes, of which 17% were high strength, 19% were moderately potent, and 36% of the isolates were with weak power (22). In a study conducted by Katongole et al. in 2020 on 200 UPEC isolates, biofilm production was reported to be 62.5% (23). The results of this study show a slight difference compared to our study, which could be due to the type of method used to measure the biofilm and differences in the sources of the sample. 
In this study, the Duplex PCR method was used to evaluate the presence of papC and sfa genes in UPEC isolates. The results showed, 15 isolates (23.4%) carried the papC gene and 10 isolates (15.6%) had the sfa gene and 9 isolates (14.06%) have both sfa and papC genes, simultaneously. In a study by Katongole et al., 22% and 13% of papC and sfa genes were reported, respectively (23). In the study of Tarchouna et al., the frequency of papC was 41% and the frequency of the sfa / foc gene was 34% (24). In a study conducted by López-Banda et al. in Mexico on 108 E. coli isolated from women with UTI, pathogenic genes, and drug resistance were examined in phylogenetic groups, and the frequency of sfaS and papC was reported to be 1.9% and 62%, respectively (25). In a study conducted by Najafi et al. in 2017 in Bushehr, Iran, on 140 UPEC isolates, the frequency of the papC gene was 38.6% and the frequency of the sfa / foc gene was 0.7% (26). The prevalence of the papC gene was 53.3% in a study by Asadi et al., in 2014 (27). In a study by Abe et al., in 225 E. coli in Brazil, they determined the frequency of pap and sfa genes to be 45.8% and 29.8%, respectively (28). A 2008 study by Grude et al. on 30 fluoroquinolone-resistant UPEC in Norway found that the frequency of pap and sfa genes was 27% and 0%, respectively (29). In the study of Tiba et al., which was performed in 2008 on 162 UPEC isolates in Brazil, the frequencies of pap, afa, and sfa genes were reported to be 32.7%, 27.8%, and 6.2%, respectively (30 ). In 2006, Arisoy et al. determined the frequency of pap and sfa genes to be 22.98% and 6.21%, respectively, by examining 161 UPEC isolates (31). In another study performed by Fathollahi et al. on E. coli, isolated from various forms of UTI, the prevalence of pap operon was reported (32). Out of 123 E. coli isolates collected from UTI by Karimian et al., 27% had pap gene and 14.6% had sfa gene (33). In Bahalo et al. study, the frequency of the pap gene was 40% and the sfa gene was 30% (34). On the other hand, in the report of Mohajeri et al., the frequency of the pap gene was 20.5% and the frequency of the sfa gene was 21.5% (35). As shown in the results of various studies, in almost all of them, the amount of pap gene is higher than sfa gene, which is consistent with the results of our research, but there is also a difference in the mentioned percentages, which may be due to differences in the number of isolates, type of samples, location of collection, the type of primer and even the protocol used for PCR. In a 2018 study by Zamani et al. on UPEC, the relationship between biofilm formation potential and genes involved in the attachment was evaluated and it was reported that there is a moderate to a strong relationship between sfaS and biofilm formation ability in biofilm-producing isolates (36). However, no significant relationship was observed between the presence of the papC gene and the ability to biofilm formation (36). Also, in the study of Katongole et al., it was found that 50% of isolates containing the papC gene were able to biofilm formation, but 53.8% of isolates containing the sfa gene were able to produce biofilms (23). The results of the present study also showed that 14 isolates (93.3%) that had the papC gene, and all (100%) isolates that had sfa gene, could produce biofilms. Also, all 9 isolates that had both studied genes simultaneously could produce biofilms. However, the results indicate the role of these genes in biofilm formation; the statistical study showed that up to the level of P-value ≤ 0.05, there was no significant relationship between antibiotic resistance and biofilm formation, as well as between the ability to form biofilm and having papC and sfa genes because there were many isolates biofilms lacked the above genes, or although antibiotic resistance was higher among isolates capable of biofilm, a significant number were sensitive to the antibiotics studied despite their ability to produce biofilms. This confirms that; although biofilm production increases antibiotic resistance in bacteria, drug resistance does not depend only on the presence of biofilm and many other factors such as the presence of degrading enzymes, the presence of effusion pumps, changes in the site of action, etc. also play a role in resistance. However, having fimbriae are effective in biofilm formation, but in addition to fimbriae, numerous other factors in bacteria (including the presence of capsules and surface proteins, etc.) are effective in biofilm formation, and these results are consistent with the results of other studies (1, 4, 6, 7, 8, 20, 22).

According to the results of this study, UPEC is still the main cause of UTI and can produce a biofilm and the binding power of this bacterium in this field has an irreversible role. On the other hand, the ability to create biofilms in 100% of isolates that have papC and sfa genes encoding fimbriae shows that fimbriae can play an effective role in this feature. The results also showed that there is a direct relationship between biofilm formation power and increased drug resistance in UPEC bacteria; Therefore, determining the pattern of E. coli antibiotic resistance in isolates obtained from UTI nationwide is very important in identifying effective drugs in the prevention and initial treatment of UTI.

This research paper is taken from the student dissertation of Master of Microbiology. The authors of this article would like to express their gratitude to the dean of the Faculty of Science and the experts of the Microbiology Research Laboratory of the Islamic Azad University, Karaj Branch.

This research has been done at personal expense and with the help and assistance of Islamic Azad University, Karaj Branch, and in the form of a master's thesis.

 

Conflicts of Interest

There is no conflict of interest between the authors of the article. All authors endorsed the final manuscript.