uropathogenic escherichia coli ( upec ) are one of the most important etiologic agents of urinary tract infection ( uti ) ( 1 , 2 ) .
uti is the most frequent human bacterial infection all around the world ( 2 ) .
it is estimated that annually global economy spends more than six billion dollars on uti ( 3 ) .
studies show that about 50% of women and 12% of men get uti during their life ( 4 ) .
also 20% to 30% of women experience recurrent infections within 6 - 12 months ( 3 , 4 )
several virulence factors such as fimbriae , toxins , and siderophores contribute to the colonization and pathogenicity of upec ( 6 ) .
virulence factors are specific traits enabling e. coli to overcome host immune system and cause various diseases ( 7 ) .
virulence genes are located on transmissible genetic elements and/or in particular regions on the chromosome that are called pathogenicity islands ( 8) .
pathogenicity islands are associated with the genome of pathogenic strains ( 9 ) and led to coordinate horizontal transfer of virulence genes between strains of one species or even related species ( 10 ) .  
upec strains also have other various types of virulence factors such as adhesins , toxins and iron uptake systems that facilitate colonization and persistence of the bacteria in the urinary tract ( 11 ) .
attachment of the bacterium to the uroepethelium is the main step to initiate and develop uti ( 12 ) .
adhesins , such as p - fimbriae , help the bacteria to resist against urinary lavage and invade epithelial cells ( 13 ) .
p - fimbriae are one of the most important adhesions encoded by pap ( pyelonephritis - associated pili ) genes and could act as predictors of pyelonephritis ( 12 )
. other important virulence characteristic of upec is serum resistance ; the ability that protects bacteria against bactericidal activity of serum ( 12 ) .
the gene trat is a surface exclusion protein that mediates resistance to serum ( 14 ) .
identification of virulence factors can be useful for diagnosis and therapeutic strategies ( 15 ) .
studies show that antibiotic resistance is increasing among upec strains every year ( 16 ) .
the high antimicrobial resistance of upec significantly reduces the therapeutic options and increases the treatment costs and mortality rates ( 17 , 18 ) . to the authors best knowledge ; few data are available regarding the virulence factors and antimicrobial resistance patterns of upec strains in iran .
the current study aimed to investigate the virulence associated determinants as well as their patterns of antibiotic resistance in upec isolated from hospitalized patients with uti .
one - hundred and fifty non - duplicate e. coli strains were isolated from urine samples of hospitalized patients with uti at shahid beheshti hospital in kashan , iran , from december 2012 to june 2013 . a positive urine culture (  10 cfu / ml ) indicated uti .
the isolates were referred to the department of microbiology , kashan university of medical sciences and identified as upec , using standard biochemical tests ( 19 ) .
all the strains were stored at -70c in tryptic soy broth ( tsb ) medium supplemented with 10% glycerol .
the antibiotic susceptibility patterns were determined using the disk diffusion method according to the clinical and laboratory standards institute ( clsi ) guidelines ( 20 ) .
the following antimicrobials were tested : amoxicillin - clavulanic acid ( amc : 20/10 g ) , ampicillin ( amp : 10 g ) , aztreonam ( atm : 30 g ) , cefoxitin ( fox : 30 g ) , ceftriaxone ( cro : 30 g ) , ciprofloxacin ( cip : 5 g ) , gentamicin ( gen : 10 g ) , nalidixic acid ( na : 30 g ) , nitrofurantoin ( nit , 300 g ) , ceftazidim ( caz : 30 g ) , trimethoprim - sulfamethoxazole ( sxt : 25 g ) and imipenem ( imp : 10 g ) .
results were interpreted as susceptible or resistant according to criteria recommended by the clsi and the manufacture protocols ( mast companies , uk ) .
bacteria were pelleted from 1.5 ml lb broth and suspended in 200 l of sterile deionized water and incubated at 100c for 10 minutes .
pcr assays were used to detect pap ( pilus associated with pyelonephritis ) , trat ( serum resistance associated ) genes and pai marker of the pathogenicity island of the upec .
the following primers were used to amplify virulence genes : 5-gcaacagcaacgctggttgcatcat-3 and 5-agagagagccactcttatacggaca-3 for pap gene to detect a 336-bp amplicon , 5- ggtgtggtgcgatgagcacag-3 and 5- cacggttcagccatccctgag-3 for trat gene to detect a 290-base pair amplicon , and 5-ggacatcctgttacagcgcgca-3 and 5 - tcgccaccaatcacagccgaac-3 for pai marker to detect a 930-bp amplicon ( 10 ) .
the virulence genes were amplified in a total volume of 25 l including 5 l of template dna , 2.5 l of 10  reaction buffer , 1 l ( 10 pmol ) of each of the forward and reverse primers , 0.5 l of 200 m of dntp , 1.5 mm mgcl2 , and 1.25 u taq dna polymerase .
pcr condition was as follows : 2 minutes at 94c and 30 cycles including denaturation at 94c for 60 seconds , annealing at 63c for 30 seconds , extension at 72c for 90 seconds and 72c for 5 minutes for the final extension in a thermal cycler apparatus ( eppendorf master cycler , ma , germany ) .
the gels were stained in ethidium bromide for 15 minutes and visualized in gel document system ( biorad , uk ) .
the sizes of the dna bands were determined by comparing them with a 100-bp dna ladder as the molecular size marker ( 100 bp dna ladder , mbi fermentas ) .
the purified pcr products were sequenced using the abi capillary system ( macrogen research , seoul , korea ) .
one - hundred and fifty non - duplicate e. coli strains were isolated from urine samples of hospitalized patients with uti at shahid beheshti hospital in kashan , iran , from december 2012 to june 2013 . a positive urine culture (  10 cfu / ml ) indicated uti .
the isolates were referred to the department of microbiology , kashan university of medical sciences and identified as upec , using standard biochemical tests ( 19 ) .
all the strains were stored at -70c in tryptic soy broth ( tsb ) medium supplemented with 10% glycerol .
the antibiotic susceptibility patterns were determined using the disk diffusion method according to the clinical and laboratory standards institute ( clsi ) guidelines ( 20 ) .
the following antimicrobials were tested : amoxicillin - clavulanic acid ( amc : 20/10 g ) , ampicillin ( amp : 10 g ) , aztreonam ( atm : 30 g ) , cefoxitin ( fox : 30 g ) , ceftriaxone ( cro : 30 g ) , ciprofloxacin ( cip : 5 g ) , gentamicin ( gen : 10 g ) , nalidixic acid ( na : 30 g ) , nitrofurantoin ( nit , 300 g ) , ceftazidim ( caz : 30 g ) , trimethoprim - sulfamethoxazole ( sxt : 25 g ) and imipenem ( imp : 10 g ) .
results were interpreted as susceptible or resistant according to criteria recommended by the clsi and the manufacture protocols ( mast companies , uk ) .
bacteria were pelleted from 1.5 ml lb broth and suspended in 200 l of sterile deionized water and incubated at 100c for 10 minutes .
pcr assays were used to detect pap ( pilus associated with pyelonephritis ) , trat ( serum resistance associated ) genes and pai marker of the pathogenicity island of the upec .
the following primers were used to amplify virulence genes : 5-gcaacagcaacgctggttgcatcat-3 and 5-agagagagccactcttatacggaca-3 for pap gene to detect a 336-bp amplicon , 5- ggtgtggtgcgatgagcacag-3 and 5- cacggttcagccatccctgag-3 for trat gene to detect a 290-base pair amplicon , and 5-ggacatcctgttacagcgcgca-3 and 5 - tcgccaccaatcacagccgaac-3 for pai marker to detect a 930-bp amplicon ( 10 ) .
the virulence genes were amplified in a total volume of 25 l including 5 l of template dna , 2.5 l of 10  reaction buffer , 1 l ( 10 pmol ) of each of the forward and reverse primers , 0.5 l of 200 m of dntp , 1.5 mm mgcl2 , and 1.25 u taq dna polymerase .
pcr condition was as follows : 2 minutes at 94c and 30 cycles including denaturation at 94c for 60 seconds , annealing at 63c for 30 seconds , extension at 72c for 90 seconds and 72c for 5 minutes for the final extension in a thermal cycler apparatus ( eppendorf master cycler , ma , germany ) .
the gels were stained in ethidium bromide for 15 minutes and visualized in gel document system ( biorad , uk ) .
the sizes of the dna bands were determined by comparing them with a 100-bp dna ladder as the molecular size marker ( 100 bp dna ladder , mbi fermentas ) .
the purified pcr products were sequenced using the abi capillary system ( macrogen research , seoul , korea ) .
a total of 150 e. coli strains were isolated from patients admitted to the various wards of shahid beheshti hospital , kashan , iran , were analyzed .
urine samples were from patients of both genders ( 78% females , 22% males ) aged between 1 - 95 years , and the mean age of 50 years .
the morphology exhibited gram - negative short rod bacteria arranged in single or paired forms .
in biochemical examination all the isolates revealed positive reaction in indole and methyl red ( mr ) tests , negative reaction in voges proskauer ( vp ) and citrate utilization tests .
of the 150 e. coli strains isolated from uti , 9 ( 6% ) were susceptible to all tested antimicrobials tested whereas 111 ( 74% ) showed resistance to more than three antimicrobial families and identified as multidrug - resistant ( mdr ) .
fifty - eight ( 38.7% ) isolates were resistant to three to six antibiotics , and 53 isolates ( 35.3% ) were resistant to more than seven antibiotics .
one isolate was resistant to imipenem and 84 ( 56% ) isolates demonstrated resistance to the extended spectrum -lactam antibiotics in disk diffusion test .
totally , the virulence genes were detected in 126 ( 84% ) upec isolates ; however in 24 strains ( 16% ) none of these virulence genes were found .
thirty - nine strains ( 26% ) had only one virulence gene , 72 strains ( 48% ) two genes ; combinations of three virulence genes were observed in 15 ( 10% ) strains .
also pcr products of pap and trat genes and pais markers after electrophoresis on agarose gels are shown in figure 1 . according to the virulence determinants , the trat gene was the most common virulence gene and was detected in 111 ( 74% ) of the isolates .
the pais markers and the pap gene were found in 92 ( 61.3% ) , and 25 ( 16.6% ) of the isolates , respectively ( table 3 ) .
the virulence associated genes were distributed in eight distinct patterns that the most common pattern was trat positive , pai positive , and pap negative ( table 4 ) .
it revealed that pcr products of 336 bp , 290 bp and 939 bp amplicons were pape allele , trat gene and pai icft073 marker , respectively .
electrophoresis of pcr product on 1.8% agarose gel ; ( a ) lanes m : 100-bp dna ladder as the molecular size marker ; lane 1 : pcr mix with no template ( negative control ) ; lane 2 : positive control for trat gene ; lanes 3 and 5- 10 : the trat gene was detected in upec strains ; lane 4 , trat -negative upec strain .
( b ) lanes m : 100-bp dna ladder as the molecular size marker ; lane 1 : pcr mix with no template ( negative control ) ; lane 2 : positive control for pap and pai genes ; lane 3 - 4 , 9 - 10 , 15 , 18 and 20 : the pap and pai negative upec strains ; lanes 5 - 6 , 8 , 11 - 12 , 16 , and 19 : pai - positive upec strains ; lane 14 : pap - positive upec strain ; lanes 7 , 13 and 17 : upec strains that were positive for both pap and pai genes .
the results of antibiotic susceptibility testing are shown in table 1 . of the 150 e. coli strains isolated from uti , 9 ( 6% ) were susceptible to all tested antimicrobials tested whereas 111 ( 74% ) showed resistance to more than three antimicrobial families and identified as multidrug - resistant ( mdr ) .
fifty - eight ( 38.7% ) isolates were resistant to three to six antibiotics , and 53 isolates ( 35.3% ) were resistant to more than seven antibiotics .
one isolate was resistant to imipenem and 84 ( 56% ) isolates demonstrated resistance to the extended spectrum -lactam antibiotics in disk diffusion test .
totally , the virulence genes were detected in 126 ( 84% ) upec isolates ; however in 24 strains ( 16% ) none of these virulence genes were found .
thirty - nine strains ( 26% ) had only one virulence gene , 72 strains ( 48% ) two genes ; combinations of three virulence genes were observed in 15 ( 10% ) strains .
also pcr products of pap and trat genes and pais markers after electrophoresis on agarose gels are shown in figure 1 . according to the virulence determinants ,
the trat gene was the most common virulence gene and was detected in 111 ( 74% ) of the isolates .
the pais markers and the pap gene were found in 92 ( 61.3% ) , and 25 ( 16.6% ) of the isolates , respectively ( table 3 ) .
the virulence associated genes were distributed in eight distinct patterns that the most common pattern was trat positive , pai positive , and pap negative ( table 4 ) .
it revealed that pcr products of 336 bp , 290 bp and 939 bp amplicons were pape allele , trat gene and pai icft073 marker , respectively .
electrophoresis of pcr product on 1.8% agarose gel ; ( a ) lanes m : 100-bp dna ladder as the molecular size marker ; lane 1 : pcr mix with no template ( negative control ) ; lane 2 : positive control for trat gene ; lanes 3 and 5- 10 : the trat gene was detected in upec strains ; lane 4 , trat -negative upec strain .
( b ) lanes m : 100-bp dna ladder as the molecular size marker ; lane 1 : pcr mix with no template ( negative control ) ; lane 2 : positive control for pap and pai genes ; lane 3 - 4 , 9 - 10 , 15 , 18 and 20 : the pap and pai negative upec strains ; lanes 5 - 6 , 8 , 11 - 12 , 16 , and 19 : pai - positive upec strains ; lane 14 : pap - positive upec strain ; lanes 7 , 13 and 17 : upec strains that were positive for both pap and pai genes .
escherichia coli is the cause of more than 80% of urinary tract infections in all age groups and can lead to renal failure if remained untreated for a long time ( 2 , 21 ) .
better knowledge of the properties of virulence and its antibiotic resistance pattern helps clinicians to anticipate the development of infection in the patients .
surface virulence factors of upec including adhesions , are responsible for colonization of bacteria in the urinary tract .
the frequency of pap gene in upec isolates in the present study was lower compared to those of found in brazil , tunisia , china , ( 5 , 6 , 22 ) , but is in agreement with the results of studies conducted by usein et al . and
santo et al . who reported the gene frequencies of 17% and 14% , respectively ( 14 , 21 ) .  
according to the literature , the frequency of pap gene can vary from 0% to 77% ( 5 , 8 , 10 , 11 ) . the diversity in frequency of pap gene among different studies
can be due to the fact that upec strains utilize a variety of adhesins to bind to the urinary epithelial cells , and start the infection .
hence , the strains lacking the pap operon may use other adhesins encoding operons such as afa , and sfa for binding .
johnson et al . ( 10 ) , reported a frequency of 71% for pai - markers among upec isolated from the patients with urosepsis in the usa . in a survey conducted by navidinia et al .
( 23 ) , a prevalence of 89% was documented for these markers in upec strains .
it is documented that the pathogenicity islands ( pais ) capable of horizontal virulence genes transfer between species ; therefore , a frequency of 61.3% for pai markers in upec strains isolated from hospitalized patients is notable .   usually , the invasive pathogens are highly resistant to the lethal activity of serum and the role of trat- protein in resistance of bacteria to serum is very important .
a study in the usa on blood samples from patients with urosepsis showed that trat is a common gene among immune - compromised patients ( 10 ) .
oliveira et al . ( 11 ) showed that 76%of the urine samples were contaminated with multidrug - resistant bacteria carrying trat gene and kudinha et al .
reported a frequency of 77% ( 2 ) for trat gene among e. coli strains isolated from patients with cystitis .
these results are compatible with those of the current study , which suggest that the trat , as a common and important virulence factor , could be considered as a target for therapeutic interventions . in the current study
, eight distinct patterns were identified among the upec strains ; whereas , high heterogeneity of the virulence genes compositions were documented in other studies ( 10 , 11 ) .
urinary tract infection is one of the most common bacterial infections and increased antibiotic resistance has complicated the treatment of such infections .
seventy - four percent of upec strains demonstrated multidrug - resistance phenotype and showed resistance to more than three of the tested antimicrobials in the present study .
high frequency of antibiotic resistance among upec strains were reported in previous studies in iran ( 8 , 24 ) .
johnson et al . ( 10 ) reported 50% multidrug - resistant e. coli among the species isolated from urine specimens .
finding of the current study showed a high rate of resistance to ampicillin , cotrimoxazole , nalidixic acid and ciprofloxacin .
studies from other countries also reported the high frequency of resistant to antibiotics in e. coli strains isolated from urine samples ( 8 , 25 - 27 ) .  
treatment failure with the first line drugs like ampicillin , cotrimoxazole , nalidixic acid has led to excessive prescription of floroquinolons and extended spectrum cephalosporins for treatment of wide range of infections including uti , gastroenteritis , lower respiratory tract infection , and other infections in iran .
floroquinolons , including ciprofloxacin , are not recommended as first - line antibiotics for the treatment of uti , but they are generally advised for the empirical therapies . regarding the results of the current study , 61.3% resistance to ciprofloxacin , which may result from discriminate use of this antibiotic in iran , is considerable . also , resistance to third generation cephalosporins , such as ceftazidim , and
( 27 ) who reported 46.4% and 55% resistance in e. coli to ceftazidime , respectively ; but are in contrast with oliveira et al .
( 11 ) findings who documented 1% ceftriaxone - resistant upec .   resistance to extended spectrum cephalosporins has increasing considerably in developing countries like iran , and it was primarily due to the ample and discriminate use of antibiotics in these countries . in the present study ,
similar results obtained from other studies ( 28 , 29 ) . in mexico , the rate of resistance to amoxicillin - clavulanic acid in e. coli strains isolated from outpatients with uncomplicated uti was 19.6% ( 28 ) . in another study on community - acquired urinary tract infections in italy
, the rate of amoxicillin - clavulanate - resistant e. coli isolates was 18.2% ( 29 ) .
because of the relatively low antibiotic resistance rate , this drug can be recommended for the treatment of urinary tract infections . according to the findings of the current study , upec strains showed high sensitivity to nitrofurantoin , which confirms the results of other studies ( 30 , 31 ) .
sensitivity to nitrofurantoin may result from decreasing the use of this drug ( 32 ) . as nitrofurantoin is unable to create appropriate level in blood flow ,
it is used only for treatment of uncomplicated utis , and not recommended for complicated uti and systemic involvements .  
the present study showed high resistance to aztreonam , which was in accordance with the findings of navidinia et al . who demonstrated that 78% of upec strains were resistant to aztreonam ( 23 ) .
high resistance to aztreonam was reported by pobiega et al . among the extended - spectrum -lactamases ( esbl)-producing e. coli strains ( 33 ) .
resistance against aztreonam may be associated with the production of esbl enzymes by esbl - producing strains , which breakdown beta - lactam ring in this antibiotic . identifying one imipenem resistant upec and four isolates with reduced susceptibility to this antibiotic were other interesting findings of the current study .
most studies have reported 100% sensitivity to imipenem ( 1 , 31 ) , but the resistance to imipenem in e. coli strains isolated from urine samples was indicated in other studies ( 34 ) .
as carbapenems are advising for the treatment of severe infections caused by esbl - producing e. coli , emerging resistance to this antibiotic is a place of major concern in treating infections caused by such resistant bacteria .   in conclusion ,
finding of the current study showed that the trat gene and pai markers were highly prevalent among upec strains isolated from hospitalized patients in kashan ; hence , they could be considered as useful targets for prophylactic interventions . also , in the current study high resistance was observed against the antibiotics widely used for the treatment of urinary tract infection ; therefore , to reach better therapeutic outcomes , empiric treatment regimens have to be modified .
 background : urinary tract infection ( uti ) is one of the most prevalent infectious diseases . 
 uropathogenic escherichia coli ( upec ) , as the most important cause of uti , are associated with a number of virulence factors.objectives:the current study aimed to investigate the virulence associated determinants as well as their patterns of antibiotic resistance in upec isolated from hospitalized patients with uti.materials and methods : a total of 150 e. coli isolates were collected from patients with uti from december 2012 to june 2013 in kashan , iran . 
 antimicrobial susceptibility screening of 12 antibiotics was determined using disk diffusion method . 
 polymerase chain reaction ( pcr ) assay was used to detect virulence - related genes in upec strains . 
 the purified pcr products were sequenced.results:of the total 150 upec isolates , 111 ( 74% ) were multidrug - resistant . 
 high resistance was observed against ampicillin ( 81.3% ) , nalidixic acid ( 71.3% ) , cotrimoxazole ( 64.7% ) and ciprofloxacin ( 61.3% ) , respectively . 
 eighty - four out of the 150 isolates showed resistance against the extended spectrum cephalosporins . 
 totally , virulence genes were detected in 126 ( 84% ) upec isolates .the pcr results identified the trat gene in ( 74% ) , pais markers in ( 61.3% ) and the pap gene in ( 16.6% ) of the isolates.conclusions:the trat gene and pai markers were highly prevalent among upec strains isolated from patients in kashan , iran ; therefore these determinants could be used as targets for prophylactic interventions . 
 also there was a high level of resistance against the antibiotics commonly used for urinary tract infection treatment . to reach better therapeutic outcomes , 
 treatment regimens have to be modified . 