Detection of uropathogenic Escherichia coli virulence factors in patients with urinary tract infections in Iran

Besides all human infectious diseases, urinary tract infections have a high prevalence and in the majority of cases Escherichia coli is a dominance bacterium which can cause pyelonephritis and cystitis. In this present study, a total of 123 strains of E. coli isolated from patients with urinary tract infections were tested in a polymerase chain reaction for detection of E. coli’s virulence factors. Results showed that fimH gene with 79.67% had the highest and tsh and usp genes respectively with 0.0% and 1.62% had the lowest presence rates of virulence genes in E. coli isolated from patients with urinary tract infections. Also 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. The significant differences (P<0.05) were seen between presence of cnf1, hlyA, pap with ompT and afa virulence genes. So, results indicated the high presence of virulence genes in patients with urinary tract infections in Iran.


INTRODUCTION
Eighty percent of urinary tract infections (UTIs) in humans are caused by Gram-negative, rod-shaped, flagellated and facultative anaerobic bacterium of the family Enterobacteriaceae with name Escherichia coli (E.coli) (Donnenberg and Welch, 1996).In addition to human, UTIs caused by E. coli has been reported from dog and cats (Féria et al., 2001).The UTIs comprise of a range of disorders, including cystitis (infection of the bladder) and pyelonephritis (infection of the kidney), which are defined by the presence of microorganisms like E. coli in urinary tract (Kulkarni et al., 2009).Foxman (2003) indicated that 40 to 50% of women have UTIs during their life.Most uropathogenic E. coli (UPEC) strains show certain virulent properties, such as adhesins, invasions, toxins, iron uptake systems and synthesis of cytotoxins and recently many novel virulence factors in UPEC strains have been introduced (Johnson, 1991).
These virulence genes help for bacterial colonization in urinary epithelium and cause severe acuity in UPEC strains which is responsible for causing severe diseases and resistance against drugs.Therefore, detection of these virulence factors is essential especially in cases with UTIs.To our knowledge, the epidemiology and  iha, ompT, irp2, cnf1, hlyA, papA, iroN, tsh, fimH and usp virulence genes of UPEC strains isolated from patients with UTIs in Iran's capital (Tehran).

Bacterial strains
A total of 123 E. coli strains isolated from patients of both sex and different ages presenting symptomatic UTIs were studied.Patients were hospitalized or visited the emergence room of Hospital Baqiyatallah (Baqiyatallah University of Medical Sciences, Tehran) in Tehran city, Iran.The strains were isolated in pure cultures and identified in the Molecular Biology Research Center at Hospital.Strains biochemically confirmed as E. coli were kept in Luria-Bertani (LB) broth /glycerol at -70°C.

DNA isolation
Bacterial strains were subcultured overnight in LB broth (Merck, Germany) and genomic DNA was extracted by using DNA extraction kit (DNP TM , CinnaGen, Iran) according to manufacturer's instruction.

Detection of urovirulence genes in E. coli
In this present study, various PCR reactions were used for detection of some virulence factors of E. coli isolate from patients with urinary tract infection in Iran.Table 1 showed the primers used for detection of UPEC virulence genes and the PCR programs including cycle, time, temperature and volumes are shown in Table 2.In all PCR reactions, for cycling, a DNA thermo-cycler (Eppendorf Mastercycler, Eppendorf-Nethel-Hinz GmbH, Hamburg, Germany) was used.The amplified products were visualized by ethidium bromide staining after gel electrophoresis of µL of the final reaction mixture in 1.5% agarose.The UPEC strains were confirmed by the gene including by detecting virulence genes by PCR method.

Statistical analysis
Statistical analysis was performed using SPSS/16.0 software for significant relationship between incidences of various urovirulence factors in E. coli isolated from patients with urinary tract infections.
Statistical significance was regarded at a P value < 0.05.

RESULTS AND DISCUSSION
After PCR on positive urine samples for the presence of E. coli in patients with UTIs, it was recognized that fimH gene with 79.67% had the highest and tsh and usp genes respectively with 0.0 and 1.62% had the lowest presence rates of virulence genes in uropathogenic E. coli (Table 3).The results showed that there were no positive samples for tsh virulence gene of E. coli isolated from patients with UTIs.Also, the prevalence of cnf1, hlyA, pap and iroN virulence factors of UPEC strains were 50.4,50.4,50.4 and 42.27%, respectively while the presence of afa, iuc, iha, ompT and irp2 were in the lower frequencies (8.13, 10.56, 17.88, 4.87 and 11.38%), respectively.The statistical analysis of data showed significant differences (P<0.05) between the presences of fimH with tsh, usp and ompT and P<0.05 between the presences of cnf1, hlyA and pap with tsh virulence genes of UPEC strains isolated from patients with UTIs in Iran.Therefore, results showed the high presences of virulence genes in E. coli isolated from patients with UTIs and excluding tsh virulence gene, other isolated E. coli strains had more than 1.5% frequency for virulence factors.
Our results indicated that the UPEC strains with this high prevalence of virulence factors can be a major causative agent for UTIs in humans in Iran.Our results showed that fimH gene with 79.67% had a highest prevalence of virulence genes in patients with UTIs but study in Romani showed that from a total 78, E. coli strains isolated from adults with different types of UTIs and the prevalence of genes coding for fimbrial adhesive systems was 86, 36, and 23% for fimH, pap, and sfa/foc, respectively and the hly and cnf genes coding for toxins were amplified in 23 and 13% of strains, respectively (Usein et al., 2001).Another study indicated that usp and iha virulence genes were present in 63.7 and 34.1% of all E. coli isolates (Bauer et al., 2002) which both were higher than our findings.Study on children with UTIs showed that the prevalence of genes, pyelonephritis Karimian et al. 6813 associated pili (pap genes), S-family adhesions (sfa gene), hemolysin (hly gene), and cytotoxic nercotizing factor type 1 (cnf1 gene) among the isolated strains was 27.1, 14.6, 13.5 and 22.9%, respectively and this study showed that Pyelonephritis was more prevalent in the cases with positive virulence genes (Farshad and Emamghorashi, 2009).Another study revealed that in addition to UTIs in humans, UPEC strains are isolated from the uterine contents in the majority of clinical cases of pyometra (Dhaliwal et al., 1998).
Study in Brazil indicated that among the E. coli isolates, the prevalence of the virulence factors were 96.0, 76.0 and 24.0%, for hemolysin, aerobactin and colicin, respectively and the prevalence of genes coding for fimbrial adhesive systems were 32.0, 19.0 and 11.0% for pap, sfa and afa respectively (Santo et al., 2006).Another study showed that a total of 12 isolates carried both pap and sfa operons, and one isolate carried both sfa and afa operons (Blanco et al., 1997) but another study showed that the simultaneous presence of pap-afa operons was not detected (Daigle et al., 1994).In spite of the previous studies, our results showed that the UPEC strains in Iran have a different virulence profile.These differences in prevalence of UPEC virulence genes showed that the virulence properties of UPEC strains is closely depending on geographic region and even weather climate of each regions.It seems that the epidemiology and prevalence of virulence factors of UPEC strains isolated from patients with UTI is different in Iran.Probably, customs, food diets, the levels of public health, hospital's health and even methods of sampling have great rules in prevalence of virulence genes of UPEC strains.In this present study, we tried to collected samples with the lowest rate of cross contamination.The significant differences about P<0.05 between the presences of fimH with tsh, usp and ompT and P<0.05 between the presences of cnf1, hlyA and pap with tsh virulence genes of UPEC strains isolated from patients with UTIs in Iran showed that surely the fimH have the prevalence of virulence factors while the tsh which was not been detected in any sample have the lowest prevalence of virulence factors in UPEC strains.On the other hand, the statistical analysis between the levels of other genes is not significant.Our results showed that the urine samples of patients with UTIs are potential sources of UPEC strains and their virulence factors.Previous study showed that from a total 161, E. coli strains isolated from children with UTIs, 22.98, 6.21, 9.94, 1.24, 9.94 and 39.75, were positive for presence of pap, sfa, afaI, hly, cnf I and aer, respectively (Arisoy et al., 2006).In addition to this high presence of virulence factors of UPEC strains isolated from patients with UTIs, usually multiple virulence factors were observed in E. coli isolates.For example in one study 44% of isolates had multiple virulence factors (Sharma et al., 2007).Another study showed that from a total of 200, E. coli 42(21%) were haemolytic, 60 (30%) showed MRHA and 72 (36%)  MSHA, 99 (49.5%) were serum resistant.In 50 controls, 8 (16%) were haemolytic, 2 (4%) showed MRHA and 4 (8%) MSHA, 16 (32%) were serum resistant (Kausar et al., 2009).

cycle: 72°C ------------5 min
Another study demonstrated that the uropathogenic E. coli strains isolated from patients with neurogenic bladder are harbouring different virulence genes implicated in the initiation and the development of the infectious process, represented by adhesins (fimH), haemolysins (hly, cnf) and iron chelating agents (aer) (Mladin et al., 2009).Another study indicated that usp and iha virulence genes were present in 63.7 and 34.1% of all E. coli isolates (Bauer et al., 2002) which both were higher than our finding.To the best of our knowledge, the presence of E. coli virulence factor is essential for the disease.On the other hand, UPEC expresses a multitude of virulence factors to break the inertia of the mucosal barrier.Some UPEC isolates, however, might possess strategies to delay or suppress the activation of components of the innate host response in the urinary tract.Despite numerous host defense mechanisms, UPEC can persist within the urinary tract and may serve as a reservoir for recurrent infections and serious complications.
Our study is the first prevalence report of direct detection of UPEC virulence factors in patients with UTIs in Iran.The results showed that PCR is an accurate assay for detection of UPEC virulence factors in patients with UTIs.

Table 1 .
Primers used for detection of virulence genes in Uropathogenic E. coli strains.

Gene Primer name Primer sequence (5'-3') Size of product (bp) References
prevalence of virulence factors of UPEC strains isolated from patients with UTIs is essentially unknown in Iran.So, this study was carried out for detection of afa, iuc,

Table 3 .
Distribution of various virulence genes of UPEC strains isolated from patients with UTIs in Iran.