Uropathogenic Escherichia coli in Iran: Serogroup distributions, virulence factors and antimicrobial resistance properties

Background Urinary tract infections (UTIs) are one of the most common bacterial infections with global expansion. These infections are predominantly caused by uropathogenic Escherichia coli (UPEC). Methods Totally, 123 strains of Escherichia coli isolated from UTIs patients, using bacterial culture method were subjected to polymerase chain reactions for detection of various O- serogroups, some urovirulence factors, antibiotic resistance genes and resistance to 13 different antibiotics. Results According to data, the distribution of O1, O2, O6, O7 and O16 serogroups were 2.43%, besides O22, O75 and O83 serogroups were 1.62%. Furthermore, the distribution of O4, O8, O15, O21 and O25 serogroups were 5.69%, 3.25%, 21.13%, 4.06% and 26.01%, respectively. Overall, the fim virulence gene had the highest (86.17%) while the usp virulence gene had the lowest distributions of virulence genes in UPEC strains isolated from UTIs patients. The vat and sen virulence genes were not detected in any UPEC strains. Totally, aadA1 (52.84%), and qnr (46.34%) were the most prevalent antibiotic resistance genes while the distribution of cat1 (15.44%), cmlA (15.44%) and dfrA1 (21.95%) were the least. Resistance to penicillin (100%) and tetracycline (73.98%) had the highest while resistance to nitrofurantoin (5.69%) and trimethoprim (16.26%) had the lowest frequencies. Conclusions This study indicated that the UPEC strains which harbored the high numbers of virulence and antibiotic resistance genes had the high ability to cause diseases that are resistant to most antibiotics. In the current situation, it seems that the administration of penicillin and tetracycline for the treatment of UTIs is vain.


Background
Urinary tract infections (UTIs) are one of the most frequent infectious diseases around the world. Urinary tract infections comprise ranges of disorders including pyelonephritis (infection of the kidney) and cystitis (infection of the bladder), which are defined by the presence of microorganisms in urinary tract [1]. Foxman (2003) indicated that 40%-50% of women have UTIs throughout their lives [2]. Also, previous report of WHO showed that the UTIs are common causes of febrile illness in 3-8% of girls and 1% of boys [3].
Escherichia coli (E. coli) is the most important cause of UTI [4,5]. Uropathogenic E. coli (UPEC) strains have shown certain virulent properties, including iron uptake systems, adhesins, specific O: K: H serotypes and synthesis of cytotoxins. All of these properties contribute to colonization and invasion of the bacterium [6]. The successful colonization of UPEC in the urinary tract depends on the expression of fimbrial adhesion proteins, which facilitate attachment of the bacterium to the urothelium, and on the presence of specific bacterial genes that encode virulence factors [7][8][9].
The treatment of diseases caused by this bacterium often requires antimicrobial therapy; however, antibiotic-resistant strains of bacteria cause more severe diseases for longer periods of time than their antibiotic-susceptible counterparts. Several studies showed that antibiotic resistance in UPEC is increasing nowadays [24,25]. Because of the high antimicrobial resistances of UPEC strains in Portugal (26%), Italy (25%) and Spain (21%) [26], the identification of bacterial resistance genes seems to be essential to reduce the treatment costs. To our knowledge, the epidemiology and prevalence of serogroups, virulence factors and antimicrobial resistance properties of UPEC strains isolated from patients with UTIs are unknown in Iran. So, the current survey was carried out in order to determine the serogroups, virulence factors and antimicrobial resistance properties of UPEC strains isolated from patients with UTIs in Iran.

Bacterial strains
A total of 123 E. coli strains isolated from patients with symptomatic UTIs were enrolled in the current investigation. The patients were hospitalized or visited the emergence room at Baqiyatallah Hospital in Tehran, Iran. The strains were isolated from pure cultures and identified in the laboratory of Molecular Biology Research Center at Hospital. The strains which were biochemically confirmed as E. coli-positive, were kept in Luria-Bertani /glycerol at −70°C.

DNA isolation
Bacterial strains were subcultured overnight in Luria-Bertani broth (Merck, Germany) and genomic DNA was extracted from typical colonies of E. coli using DNA extraction kit (DNP ™ , CinnaGen, Iran) according to manufacturer's instruction.

Detection of uropathogenic E. coli serogroups, virulence factors and antibiotic resistance genes
In the present study various PCR assays were used for detection of serogroups, virulence factors and antimicrobial resistance genes of UPEC E. coli isolates. Table 1 showes the primers applied for detection of UPEC virulence genes, Table 2 indicates the primers used for detection of UPEC serogroups and finally in Table 3 the primers used for detection of antimicrobial resistant genes in uropathogenic E. coli are shown. The amplified products were visualized by ethidium bromide staining after gel electrophoresis of 10 μL of the final reaction mixture in 1.5% agarose.

Antimicrobial susceptibility testing
Antimicrobial susceptibility tests was performed by the Kirby-Bauer disc diffusion method using Mueller-Hinton agar (HiMedia Laboratories, Mumbai, India, MV1084), according to the Clinical and Laboratory Standards Institute guidelines [40]. After incubating the inoculated plates aerobically at 37°C for 18-24 h in an aerobic atmosphere, the susceptibility of the E. coli isolates to each antimicrobial agent was measured and the results were interpreted in accordance with interpretive criteria provided by CLSI (2006). E. coli ATCC 25922 was used as quality control organisms in antimicrobial susceptibility determination.

Statistical analysis
The data were analyzed using SPSS software (Version 17. SPSS Inc, United States) to find any significant correlation between incidences of virulence factors and antibiotics resistance genes of uropathogenic E. coli serogroups isolated from patients with urinary tract infection. Statistical significance was regarded at a P value < 0.05.

Ethical issues
In the current study we tried to protect the life, health, dignity, integrity, rights to self-determination, privacy, and confidentiality of personal information of research subjects. We conform to generally accepted scientific principles, be based on a thorough knowledge of the scientific literature, other relevant sources of information, and adequate laboratory and, as appropriate, animal experimentation. All samples were taken from volunteer patients for this research. All ethical issues were considered and this research was performed with hospitals' permission. The name and characters, personal information and even patients' illnesses and their medical information remained secret. In addition, in this cooperation agreement we stated that this research will help urology and microbiology and is able to clarify the epidemiology Irp2-F AAGGATTCGCTGTTACCGGAC 413 [34] Irp2-R AACTCCTGATACAGGTGGC and prevalence of stereotypes, virulence factors and antimicrobial resistance of UPEC strains isolated from patients with urinary tract infection. All of the patients showed their satisfaction in order to use their sample in this investigation especially to determine antibiotic resistance in UPEC strains.

Results and discussion
Our results revealed high distribution of UPEC serogroups isolated from patients with urinary tract infection (   (Table 4). Besides, the serogroups of 13.82% UPEC strains isolated from these patients could not be detected and were diagnosed as non-detected serogroups (Table 4).
Our results revealed that the UPEC strains were able to be one of the major causative agents of UTIs in Iran and this finding was in accordance with the previous study which mentioned that 150 million people are diagnosed as UTI-positive annually [41]. Also, The UPEC strains are isolated from the uterine contents in the majority (82-100%) of clinical pyometra cases [42,43]. Therefore, such a high prevalence of serogroups, virulence factors and antibiotic resistance genes in UPEC strains exhibits that there is a high risk of developing incurable diseases.
Several investigations have been performed on the prevalence of UPEC strains in UTI cases in Iran [44,45]. Kalantar et al. [44] showed that the E. coli was the most frequent occurring pathogen (54.80%) in patients with severe UTIs. Esmaeili [45] demonstrated that the E. coli was the most common cause of UTI in human. Also, previous study has been reported that the incidence of UTI has been increased recently [46].
Based on our results, there were significant differences about (P < 0.01) between the presence of fim, tsh and sen virulence genes as well as set1, tsh and sen genes (P < 0.05) and also between fim and usp genes of isolated E. coli strains (P < 0.05). Therefore, fim and set1 were the most common virulence genes. Arabi et al. [54] showed the similar results of UPEC virulence genes in Iran. Arabi et al. [54] indicated that the fim and sfa fimbriae genes were observed in 92.7% of isolates, separately. Also, Asadi Karam et al. [55] showed that the fim genes were the most prevalent virulence genes of UPES strains. Karimian et al. [56] proved that fimH gene with the frequency rate of 79.67% was the most and tsh and usp genes with the frequency rate of 0.0% and 1.62% respectively were the least common virulence genes in E. coli strains isolated from patients with urinary tract infections. Also, Karimian et al. [56] showed that the presence of cnf1, hlyA, pap, iroN, afa, iuc, iha, ompT and irp2 virulence genes were 50.4, 50.4, 50.4, 42.27, 8.13, 10.56, 17.88, 4.87 and 11.38%, respectively. Other virulence genes of UPEC strains like aatA, aggR and stbA [47] and stx1 and stx2 [57] have been reported from Iran previously. Another investigation announced that the prevalence rate of fimH, fyuA, kpsMTII and iucD genes were above 75% likewise papC, papG, sat, iron, usp and traT were between 35-65% [12].
Our results contrary to other studies indicated that there are the possibilities of the existence of multiple virulence genes in UPEC strains isolated from UTIs patients [58,59]. The importance of UPEC strains' sfa gene in patients with severe UTIs has been reported previously [59,60] while Abe et al. [11] and Santo et al. [59] reported lower prevalence of sfa gene among UPEC. Another study indicated that usp and iha virulence genes were present in 63.7% and 34.1% of all E. coli isolates [22] which both were higher than our findings.
The statistical analyses were significant among aadA1, cat1 and cmlA antibiotic resistance genes (P < 0.05). This correlation was demonstrated among tetracycline and nitrofuration (P < 0.05) and also penicillin, trimethoprim and gentamycin (P < 0.05) too. High frequency of resistant UPEC strains to one or more antimicrobials was observed in the present work and it was in agreement with previous studies [61,62].
Recently, trimethoprim-sulfamethoxazole was used as a standard antibiotic for a calculated UTIs therapy and due to the increased resistance of UPEC strains to this class of antibiotics, fluoroquinolones as broad-spectrum antimicrobial agents have been used with increasing frequency in complicated as well as uncomplicated UTIs [66], but after a short time resistance to fluoroquinolones was emerged [67]. Previous study showed that more than 10% of the E. coli isolated in 2000-2002 from intensive care unit patients in European and North American were resistant to ciprofloxacin [68], while this amount of resistance was increased significantly in our evaluation.
Gulsun et al. [69] reported that the sensitivity of the UPEC strains to the norfloxacin, ciprofloxacin, netilmicin, amikacin, ceftriaxone, gentamicin, nitrofurantoin, amoxicillin-clavulanate, trimethoprim/sulfamethoxazole and ampicillin were 89%, 85%, 80%, 78%, 74%, 72%, 71%, 58%, 45%, 35%, respectively which was somewhat similar to our progeny. In a study carried out in India, the highest resistance have been shown against amoxicillin (67.3%) and least against nitrofurantoin (57.3%) [70] which was similar to our findings. On the other hand our results revealed that in the current situations the nitrofurantoin is a choice drug due its lowest antibiotic resistance in UPEC strains. Eighty five to ninety two percent of UPEC strains were sensitive to nitrofurantoin in previous study [53].
Our results showed that resistance to chloramphenicol was 25.5% but chloramphenicole is a forbidden antibiotic and the high antibiotic resistance to this drug in our study indicated the irregular and unauthorized use of this drug in medicine treatments. Unfortunately, not only in medicine, but also veterinarians in many fields of veterinary such as large animal internal medicine, poultry and even aquaculture, use this antibiotic as a basic one. Therefore, in a very short period of time, antibiotic resistance will appear.

Conclusions
Based on our results, O25 serotype, fim virulence gene, aadA1 antibiotic resistance gene and finally resistance to penicillin had highest frequencies in UPEC strains isolated from UTIs patients. The results of our study revealed the high presence of UPEC strains in patients with UTIs in Iran. To our knowledge, our study is the first report of direct detection of serogroups, virulence factors and antimicrobial resistance properties of uropathogenic E. coli strains in Iran. Due to the high prescription of antibiotics in humans and even animals, antibiotics resistance has been increased in UPEC strains and we recommend using antibiotics only in severe conditions and applying strong antibiotics and multi antibiotic descriptions.