specimens and culture process : four hundred and ninety stool samples were 
 collected from calves with diarrhea during a 1-year period from a major farm in tehran , 
 iran . each swab sample was inoculated to amies transport media with charcoal ( merck kgaa , 
 darmstadt , germany ) and streaked on macconkey agar ( merck kgaa ) within 4 hr of collection . 
 up to 3 suspected colonies on macconkey agar were selected and subjected to biochemical 
 tests including gram staining , oxidase , indol , simmons citrate , urease and hydrogen sulfide 
 as described previously .
the biochemically 
 confirmed e. coli colonies were subjected to molecular analysis to search 
 for the enterohemorrhagic pathotype .
molecular diagnostic methods for ehec and sequence analysis of integron 
 contents : genomic dna was extracted from purified e. coli 
 cultures using the boiling method .
accordingly , bacterial cells were pelleted by 
 centrifugation and suspended in 50 l of te buffer and subsequently 
 subjected to boiling for 10 min at 100c .
the lysate was centrifuged , and the supernatant 
 was subjected to pcr assay using 3 different sets of primers targeting the 
 stx1 , stx2 and eae genes .
class 1 
 integron was detected among confirmed ehec isolates using primers that specifically detect 
 the conserved integrase gene ( intf / int - r ) .
the resistance gene cassette within the variable 
 region of class 1 integron was further characterized using primers that spans between the 
 3-cs and 5-cs conserved regions of integron and amplifies the entire gene cassette 
 integrated ( in - f / in - r ) .
the primer sequences used in this study are shown in table 1table 1.primers sequences used in this studyprimersequence ( 5-3)amplicon size ( bp)referenceeaetgcggcacaacaggcggcga42228cggtcgccgcaccaggattcstec / stx1cagttaatgtggtggcgaag8945ctgctaatagttctgcgcatcstec / stx2cttcggtatcctattcccgg4785ggatgcatctctggtcattginttgcgtgtaaatcatcgtcgt9002caaggttctggacagttgcinggcatccaagcagcaagcvariable9aagcagacttgacctgat .
pcr was performed in a reaction mixture with a total volume of 25 
 l containing 5 l template dna ( 20 
 ng ) , 2.5 l 10x taq polymerase buffer [ 100 mm tris / hcl ( ph 
 8.3 ) , 500 mm kcl and 15 mm mgcl2 ] , 0.25 l ( 100 
 pmol/l ) each of primers , 0.25 l dntps ( 10 mm ) , 0.2 
 l ( 5 u/l ) taq dna polymerase and 16.8 
 l sterilized distilled water .
amplification was done as follows : an initial denaturation step was 
 performed at 94c for 5 min followed by 30 cycles consisting of denaturation ( 94c for 1 
 min ) , annealing ( 58c for 1 min , separately adjusted for each set of primer pairs ) and 
 extension ( 72c for 1 min ) , and this was followed by a final extension step at 72c for 3 
 min . each pcr product from 3-cs/5-cs amplification was purified and directly sequenced 
 ( macrogen research , seoul , korea ) and compared with the prototype strain salmonella 
 enterica serotype typhimurium ( accession number ab365868.1 ) as suggested 
 previously .
antimicrobial susceptibility testing : antimicrobial susceptibility of 
 isolates was examined using the disk diffusion method on mueller 
each isolate was tested for susceptibility to 7 antimicrobial agents : 
 ampicillin ( ap ) ( 10 mg ) , chloramphenicol ( c ) ( 10 mg ) and streptomycin ( st ) ( 10 mg ) from 
 becton dickinson and company ( sparks , md , u.s.a . ) , and ciprofloxacin ( cp ) ( 1 mg ) , gentamicin 
 ( gm ) ( 10 mg ) , trimethoprim ( tr ) ( 2/5 mg ) and tetracycline ( te ) ( 10 mg ) from difco 
 laboratories ( detroit , mi , u.s.a . ) .
genbank accession numbers : one representative of each group of integrated 
 gene cassettes with identical nucleotide sequences was deposited in the genbank database . 

pulsed - field gel electrophoresis : a pulsenet standardized protocol 
 described for subtyping of escherichia spp . was used for genotyping . in brief ,
bacterial plate cultures were suspended in 
 a cell suspension buffer ( 100 mmol l tris , 100 mmol l edta , ph 
 8.0 ) and adjusted to absorbance values of 0.8 to 1.0 at a wavelength of 610 nm and used for 
 preparation of agarose plugs with seakem gold agarose ( lonza , rockland , me , u.s.a . ) and 
 proteinase k. after a lysis step with 50 mmol l tris , 50 mmol l 
 edta ( ph 8.0 ) , 1% sarcosine , and 0.5 mg of proteinase k , followed by washing steps , 40 units 
 of xbai restriction enzyme ( roche diagnostic deutschland gmbh , mannheim , 
 germany ) was used for digestion of plug - embedded dnas .
a dna molecular weight size marker 
 was prepared by xbai digestion of salmonella enterica 
 serotype braenderup h9812 plugs
. a chef mapper xa system ( bio - rad ) was applied for 
 electrophoresis with 200 v at 14c for 18 hr with the pulsed time increasing from 2.16 sec 
 to 54.17 sec .
gelcompar ii version 4.0 software ( applied maths , sint - martens - latem , belgium ) 
 was used for analysis of pfge - generated patterns using the dice coefficient and upgma 
 ( unweighted pair group method with arithmetic averages ) clustering .
a dendrogram was 
 constructed with an optimization value of 0.50% and a position tolerance of 1.0% .
a total of 21 ( 4.3% ) enterohemorrhagic e. coli strains were identified in 
 this study with the eaestx1stx2 
 genotype .
amplification bands of 422 bp , 894 bp and 478 bp were obtained for 
 eae , stx1 and stx2 , respectively .
the 
 results of pcr analyses for each pair of primer sets are depicted in fig .
b ) pcr 
 amplification of the eae gene ( lanes 1 - 4 ) .
c ) pcr amplification of 
 the class 1 integron integrase gene ( int ) ; lanes 1 - 6 , isolates with 
 class 1 integron ; lane 7 , negative control .
lanes 1 and 2 , 700 bp gene cassettes ; lanes 3 and 
 4 , 700/1,700 bp gene cassettes ; lanes 5 and 6 , 1,700 bp gene cassettes .
b ) pcr 
 amplification of the eae gene ( lanes 1 - 4 ) .
c ) pcr amplification of 
 the class 1 integron integrase gene ( int ) ; lanes 1 - 6 , isolates with 
 class 1 integron ; lane 7 , negative control .
lanes 1 and 2 , 700 bp gene cassettes ; lanes 3 and 
 4 , 700/1,700 bp gene cassettes ; lanes 5 and 6 , 1,700 bp gene cassettes .
all of the strains showed resistance to am , cp , tr , st , c and te , and 19% showed resistance 
 to gm .
distribution of class 1 integron : out of 21 ehec strains , 11 ( 53% ) 
 harbored an integrase gene specific for class 1 integron 
 ( int1 ) using primers int1-f and int1-r 
 ( 5 conserved region ) with a dna fragment of 900 bp .
two different amplification products , 
 which were approximately 750 and 1,700 bp in size , were obtained from amplified variable 
 regions ( in - f / in - r primers ) in 3 ( 14.3% ) and 4 ( 19% ) of 
 the ehec isolates .
the amplified fragments corresponded to dfra7 
 ( dihydrofolate reductase type i ) and dfra1/aada1 
 ( dihydrofolate reductase / aminoglycoside adenyltransferase ) resistance gene cassettes , 
 respectively , which was confirmed by sequencing . both of the amplification bands ( 750 and 
 1,700 bp )
were simultaneously observed in 2 ( 9.5% ) of the isolates indicative of the two 
 gene cassettes .
no product was produced from 2 int isolates , 
 suggesting an empty integron with no resistance gene cassette ( fig .
were compared using the dice 
 coefficient and upgma analysis , and the data revealed a total of 16 pulsotypes , which 
 corresponded to 16 isolates , 9 of which were harboring class 1 integron .
analysis of 
 pulsotypes showed extensive diversity among the isolates harboring integron , which is 
 indicative of a lack of any significant genetic relatedness among the isolates .
similarity 
 indices of 62% and 30% were obtained for the most and the least similar isolates , 
 respectively ( fig .
the isolates are presented in 
 comparison with their phenotypic resistance profiles and integron resistance gene 
 contents . ) .
of the 21 isolates , 4 ( 19% ) did not produce any restriction pattern , and their dna 
 repeatedly appeared as smears on the gel , even after addition of thiourea to the running 
 buffer .
the isolates are presented in 
 comparison with their phenotypic resistance profiles and integron resistance gene 
 contents .
in pathogen detection systems before 2011 , e. coli patho - types , notably 
 epec and ehec isolates , had been identified by serotyping based on their o - group 
 specificities , which was increasingly problematic due to the incapability of this method to 
 clearly separate the pathotypes .
therefore , different molecular methods have been developed 
 to more accurately assign the isolates to each pathotype based on their virulence - associated 
 genes [ 5 , 28 ] . 

accordingly , the eae , stx1 and stx2 genes 
 were used in this study for molecular identification of isolates .
all of the strains under study were resistant to all tested antimicrobial agents including 
 am , cp , tr , st , c and te . the most commonly used antibiotics in conventional dairies are 
 penicillin and tetracyclines [ 11 , 37 ] .
this may correlate with the resistance of all 
 isolates to these two groups of antibiotics .
our previously published data indicated the 
 same resistance profile among enteropathogenic e. coli strains isolated 
 from children with diarrhea .
a high level of 
 resistance to different classes of antibiotics among enterohemorrhagic and enteropathogenic 
 pathotypes of e. coli suggests the involvement of the same mechanisms in 
 emergence of resistance in the two pathotypes ; moreover , the circumstances of the intestinal 
 environment in the acquisition of different mobile elements including integrons , 
 transposons , etc , should not be ignored .
distribution of class 1 integrons among members of the enterobactericeae family is likely 
 to be common around the world , and it has been shown to be associated with a variety of 
 resistance gene cassettes , the encode for resistance to different antibiotics .
a total of 53% of our ehec isolates harbored class 1 
 integron , and the most common integrated resistance gene cassettes belonged to the 
 dfr and/or aada family .
the coexistence of these 2 
 families of resistance gene cassettes among human clinical isolates of 
 shigella spp .
, salmonella enterica and 
 escherichia coli was previously reported by our laboratory [ 12 , 28 ] .
this 
 emphasizes the wide distribution of these 2 gene cassette families among clinical and 
 veterinary enteric bacteria , which provides evidence for horizontal gene transfer in the 
 environment or intestinal tract of humans or animals ; however , more studies are required to 
 understand the exact mechanisms involved .
guerra et al . reported the presence of class 1 integron in 41% of 105 ehec 
 isolates from humans with enteric disease ( 97 isolates ) and cattle ( 8 isolates ) that carried 
 dfra15-aada1 , dfra1-aada1 , aada1 or no 
 gene cassettes in their variable regions .
cergole - novella et al . reported the presence of class 1 integron in 22% of 
 antimicrobial resistant shiga - toxin - producing e.coli isolated from patients 
 with diarrhea
( 21 isolates ) and cattle ( 11 isolates ) that harbored identical single gene 
 cassettes ( aada1 ) in their variable regions .
reported that the prevalence of class 1 integrons was 
 14% in enterohemorrhagic e.coli isolates from humans and animals and that 
 the isolates uniformly contained the aada gene .
these reports together with our findings suggest the worldwide distribution of the 
 aada and dfra aminoglycoside resistance gene family 
 among integrons , which further emphasizes the need to monitor their contents for fear of 
 acquisition of resistance genes against newer antimicrobial agents .
genotyping analysis revealed extensive heterogeneity among the isolates , which indicates a 
 diverse population of ehec in the intestinal tract of cattle .
this finding is in contrast 
 with genotyping studies on ehec strains isolated from cattle and humans from japan , which 
 have shown a high degree of homogeneity among their isolates .
changes in the pfge pattern of xbai - digested dna 
 fragments of ehec isolates have been shown in association with loss of shiga toxin genes 
 during maintenance or subcultivation , which is related to disappearance of a 70- or 80-kb 
 fragment .
nevertheless , little disparity has 
 been reported in the pfge pattern of ehec isolates of different origins .
isolates with identical integron contents showed different genotyping patterns , and no 
 clear - cut relationship was obtained between the specific pulsotypes and their integron 
 contents .
this highlights the potential of integrons for integration within different ehec 
 isolates regardless of their genotypes .
the antimicrobial resistance profiles were identical 
 among the isolates , except for susceptibility to gentamicin .
diversity in the resistance 
 profile of isolates was also not reflected in their genotype properties .
overall , these data 
 suggest that the genotyping patterns of isolates are independent of their resistance 
 properties and integron contents . to our knowledge ,
little data exist on the genotyping patterns of ehec isolates from cattle 
 in relation to their resistance gene contents , which signifies the need for this to be 
 worked on .
this emphasizes the need for establishment of national programs in iran and in 
 the middle east and exchange of genotyping databases in order to monitor and source track 
 the source of emergence and spread of new resistant and integron - carrying genotypes within 
 these areas .
 abstracta total of 21 ( 4.3% ) 
 enterohemorrhagic e. coli strains were isolated by biochemical tests and 
 identification of the eae+stx1+stx2 + 
 genotype from 490 stool samples obtained from calves with diarrhea during 1-year period 
 from a major farm in tehran , iran . 
 all of the strains showed resistance to ampicillin , 
 ciprofloxacin , trimethoprim , streptomycin , chloramphenicol and tetracycline , while 19% 
 showed resistance to gentamicin . 
 out of 21 ehec strains , 11 ( 53% ) harbored class 1 
 integron . 
 two different amplification products , which were approximately 750 and 1,700 bp 
 in size , were obtained from amplified variable regions 
 ( in - f / in - r primers ) in 3 ( 14.3% ) and 4 ( 19% ) of the 
 ehec isolates , which corresponded to dfra7(dihydrofolate reductase type 
 i ) and dfra1/aada1(dihydrofolate 
 reductase / aminoglycoside adenyltransferase ) resistance gene cassettes , respectively , and 
 this was confirmed by sequencing . 
 genotyping analysis revealed a total of 16 pulsotypes 
 that corresponded to 16 isolates with the similarity indices of 62% and 30% for the most 
 and least similar isolates , respectively , 9 of which harbored class 1 integron . 
 analysis 
 of pulsotypes showed an extensive diversity among the isolates harboring integron , which 
 is indicative of a lack of any significant genetic relatedness among the isolates . 
 no 
 obvious relation could be deduced between integron content and special pulsotypes . 
 the 
 little data available on the genotyping patterns of ehec isolates from cattle and their 
 resistance gene contents emphasize the need to establish genotyping databases in order to 
 monitor and source track the source of emergence and spread of new resistant and 
 integron - carrying genotypes . 
MATERIAL AND METHODS
RESULTS
DISCUSSION