BACKGROUND
enteropathogenic escherichia coli  are an important cause of infant diarrhea in developing countries
<cit> .
the majority of epec isolates belong to classic serotypes derived from  <dig> classical o serogroups
<cit> .
epec induces attaching and effacing  lesions on epithelial cells, characterized by microvilli destruction, cytoskeleton rearrangement, and the formation of a pedestal-like structure at the site of bacterial contact
<cit> .
the a/e genes are localized to the locus for enterocyte effacement  and encode intimin, a type iii secretion system, secreted proteins and the translocated intimin receptor
<cit> .
“typical” epec strains  contain also the epec adherence factor  plasmid
<cit> , which carries genes encoding a regulator
<cit>  and the bundle-forming pili
<cit> .
epec strains lacking the eaf plasmid have been designated “atypical” epec
<cit> .
recent epidemiological studies indicate that aepec are more prevalent than tepec in both developed and developing countries
<cit> .
some aepec strains are genetically related to the enterohemorrhagic e. coli , and both are considered as emerging pathogens
<cit> .
typical epec strains express only the virulence factors encoded by the lee region and the eaf plasmid, with the exception of the cytolethal distending toxin produced by o86:h <dig> strains and the enteroaggregative heat-stable enterotoxin  <dig>  found in o55:h <dig> and o127:h <dig> strains.
in contrast, aepec strains frequently express east <dig> and additional virulence factors not encoded by lee region
<cit> .
in a previous study
<cit> , east <dig> was the most frequent  virulence factor found in a collection of  <dig> aepec strains, and was significantly associated with children diarrhea.
east1-positive aepec strains have been associated with outbreaks of diarrhea involving children and adults in the united state
<cit>  and japan
<cit> .
however, it is not sufficient to simply probe strains with an asta gene probe due to the existence of east <dig> variants
<cit> .
in one study, 100% of the o <dig>  o <dig>  o <dig>  and o157:h <dig> enterohemorrhagic e. coli  strains examined carried dna sequences homologous to the east <dig> gene  with two different mutation types.
type  <dig> sheast has  <dig> nucleotide non-synonymous substitutions including one in the initiation codon; type  <dig> sheast lacks the first  <dig> codons of east <dig> sequence
<cit> .
the focus of the study was to investigate the asta gene sequence present in tepec and aepec strains.
the strains were collected in different cities of brazil in different periods of time and in a previous study poor relatedness was observed by rapd analysis of  <dig> strains belonging to this collection
<cit> .
RESULTS
we examined  <dig> epec strains  for the presence of the asta gene by pcr using primers that anneal to the 5’ ends of the eaec  <dig> asta gene sequence
<cit> .
those strains were isolated from diarrheic and non diarrheic brazilian children in previous studies
<cit> .
as shown in table
<dig>   <dig>  tepec and  <dig>  aepec strains were positive in the pcr assay.
among the aepec pcr-positive strains,  <dig> belonged to the o <dig> and o <dig> serogroups.
asta
strains isolated from diarrheic and non-diarrheic children
ao2:h2;h45; o101:h33; o145:hnd; o157:hnd; o162:h33; ont:h45; ont:hnd.
bo4:hnd; o15:hnd o33:h6; o35:h19; o37:hnd; o49:hnd; o61:hnd; o63:hnd; o79:hnd; o85:h40; o96:hnd; o98:hnd; o101:nm; o103:nm; o105:h7; o108:h31; o109:h54; o117:hnd; o132:hnd; o141:hnd; o1523h2; o156:h16; o157:hnd; o167:h6; o169:h6; o175:hnd;ont:nm; ont:h18; ont:hnd.
note: nm, nommotile, nd, nondetermined, ont, nontypeable.
the  <dig> asta gene pcr products were sequenced.
twenty five strains,  <dig> tepec and  <dig> aepec, carried the dna sequence identical to the east <dig> gene  .
a subgroup of  <dig> aepec strains presented a variant type of the 042-type east <dig> gene sequence, with four non-synonymous nucleotide substitutions.
nine other strains, including one typical, carried either the sequence identical to type  <dig> sheast  or to type  <dig> sheast .
the remaining  <dig> strains carried mutated sequences of the 042-type east <dig> , type  <dig> sheast  or type  <dig> sheast  genes.
the expression of east <dig> was examined by rt-pcr and quantitative rt-pcr.
the rt-pcr results showed that the asta gene was transcribed only by the strains carrying either the intact or the variant type of the asta gene sequence .
the asta gene expression levels of the  <dig> rt-pcr positive strains  were nearly identical to that of eaec  <dig> strain .
plasmids of the  <dig> pcr-positive strains were examined for asta gene presence by southern blot hybridization with the asta probe.
in  <dig>  strains, a single copy of the asta gene was located to a large plasmid .
in all the eleven tepec strains, the asta probe hybridized to the eaf plasmid as previously reported
<cit> , and in twelve aepec the asta probe hybridized with large plasmids of similar size.
the plasmids of the remaining strains were asta probe negative.
we previously reported that 24% of  <dig> aepec strains hybridized with a dna probe for east1
<cit> .
here, we analyzed by pcr a larger group of epec, including typical strains and found that  <dig>  of  <dig> tepec and  <dig>  of  <dig> aepec were asta positive.
sequence analysis of the pcr products showed that  <dig>  of  <dig> tepec and  <dig>  of  <dig> aepec had an intact 042-type asta gene.
as shown in table
<dig>  strains carrying intact asta gene were more frequently found in diarrheic children than in non-diarrheic children .
however, we should point out that among the  <dig> strains analyzed only  <dig> were collected from a case–control study
<cit> .
asta
gene found in epec strains isolated from diarrheic and non-diarrheic children
asta
ap =  <dig> ; fisher’s exact test .
the east <dig> gene family includes one major type of sequence, i.e.
the asta of eaec strain  <dig> that is widely distributed among different diarrheagenic e. coli strains
<cit>  and four variant types of east <dig>  i.e.
the east1v <dig> of eaec 17–2
<cit> , east1v <dig> of epec n1
<cit> , and east1v <dig> and east1v <dig> of e. coli o166:h15
<cit> .in this study, a subgroup of aepec strains had a new variant type of east <dig> gene sequence that differed from those previously reported, and was denominated east1v <dig> .
the rt-pcr analysis showed that east1v <dig> was transcribed to produce mrna.
however, more studies are necessary to determine whether east1v <dig> is associated with a functional polypeptide toxin.
CONCLUSIONS
in conclusion, our data suggest that the presence of an intact asta gene may represent an additional virulence determinant in both epec groups.

 enteropathogenic escherichia coli are an important cause of infant diarrhea in developing countries. the majority of epec isolates belong to classic serotypes derived from classical o serogroups. epec induces attaching and effacing lesions on epithelial cells, characterized by microvilli destruction, cytoskeleton rearrangement, and the formation of a pedestal-like structure at the site of bacterial contact. the a/e genes are localized to the locus for enterocyte effacement and encode intimin, a type iii secretion system, secreted proteins and the translocated intimin receptor. “typical” epec strains contain also the epec adherence factor plasmid, which carries genes encoding a regulator and the bundle-forming pili. epec strains lacking the eaf plasmid have been designated “atypical” epec. recent epidemiological studies indicate that aepec are more prevalent than tepec in both developed and developing countries. some aepec strains are genetically related to the enterohemorrhagic e. coli, and both are considered as emerging pathogens. typical epec strains express only the virulence factors encoded by the lee region and the eaf plasmid, with the exception of the cytolethal distending toxin produced by o86:h strains and the enteroaggregative heat-stable enterotoxin found in o55:h and o127:h strains. in contrast, aepec strains frequently express east and additional virulence factors not encoded by lee region. in a previous study, east was the most frequent virulence factor found in a collection of aepec strains, and was significantly associated with children diarrhea. east1-positive aepec strains have been associated with outbreaks of diarrhea involving children and adults in the united state and japan. however, it is not sufficient to simply probe strains with an asta gene probe due to the existence of east variants. in one study, 100% of the o o o and o157:h enterohemorrhagic e. coli strains examined carried dna sequences homologous to the east gene with two different mutation types. type sheast has nucleotide non-synonymous substitutions including one in the initiation codon; type sheast lacks the first codons of east sequence. the focus of the study was to investigate the asta gene sequence present in tepec and aepec strains. the strains were collected in different cities of brazil in different periods of time and in a previous study poor relatedness was observed by rapd analysis of strains belonging to this collection.  we examined epec strains for the presence of the asta gene by pcr using primers that anneal to the 5’ ends of the eaec asta gene sequence. those strains were isolated from diarrheic and non diarrheic brazilian children in previous studies. as shown in table  tepec and aepec strains were positive in the pcr assay. among the aepec pcr-positive strains, belonged to the o and o serogroups. asta strains isolated from diarrheic and non-diarrheic children ao2:h2;h45; o101:h33; o145:hnd; o157:hnd; o162:h33; ont:h45; ont:hnd. bo4:hnd; o15:hnd o33:h6; o35:h19; o37:hnd; o49:hnd; o61:hnd; o63:hnd; o79:hnd; o85:h40; o96:hnd; o98:hnd; o101:nm; o103:nm; o105:h7; o108:h31; o109:h54; o117:hnd; o132:hnd; o141:hnd; o1523h2; o156:h16; o157:hnd; o167:h6; o169:h6; o175:hnd;ont:nm; ont:h18; ont:hnd. note: nm, nommotile, nd, nondetermined, ont, nontypeable. the asta gene pcr products were sequenced. twenty five strains, tepec and aepec, carried the dna sequence identical to the east gene. a subgroup of aepec strains presented a variant type of the 042-type east gene sequence, with four non-synonymous nucleotide substitutions. nine other strains, including one typical, carried either the sequence identical to type sheast or to type sheast. the remaining strains carried mutated sequences of the 042-type east, type sheast or type sheast genes. the expression of east was examined by rt-pcr and quantitative rt-pcr. the rt-pcr results showed that the asta gene was transcribed only by the strains carrying either the intact or the variant type of the asta gene sequence. the asta gene expression levels of the rt-pcr positive strains were nearly identical to that of eaec strain. plasmids of the pcr-positive strains were examined for asta gene presence by southern blot hybridization with the asta probe. in strains, a single copy of the asta gene was located to a large plasmid. in all the eleven tepec strains, the asta probe hybridized to the eaf plasmid as previously reported, and in twelve aepec the asta probe hybridized with large plasmids of similar size. the plasmids of the remaining strains were asta probe negative. we previously reported that 24% of aepec strains hybridized with a dna probe for east1. here, we analyzed by pcr a larger group of epec, including typical strains and found that of tepec and of aepec were asta positive. sequence analysis of the pcr products showed that of tepec and of aepec had an intact 042-type asta gene. as shown in table  strains carrying intact asta gene were more frequently found in diarrheic children than in non-diarrheic children. however, we should point out that among the strains analyzed only were collected from a case–control study. asta gene found in epec strains isolated from diarrheic and non-diarrheic children asta ap = ; fisher’s exact test. the east gene family includes one major type of sequence, i.e. the asta of eaec strain that is widely distributed among different diarrheagenic e. coli strains and four variant types of east i.e. the east1v of eaec 17–2, east1v of epec n1, and east1v and east1v of e. coli o166:h15.in this study, a subgroup of aepec strains had a new variant type of east gene sequence that differed from those previously reported, and was denominated east1v. the rt-pcr analysis showed that east1v was transcribed to produce mrna. however, more studies are necessary to determine whether east1v is associated with a functional polypeptide toxin.  in conclusion, our data suggest that the presence of an intact asta gene may represent an additional virulence determinant in both epec groups. 
a subgroup of atypical strains had a variant type of the asta gene sequence, with four non-synonymous nucleotide substitutions. the rt-pcr results showed that the asta gene was transcribed only by the strains carrying either the intact or the variant type of the asta gene sequence. strains carrying intact asta genes were more frequently found in diarrheic children than in non-diarrheic children. in conclusion, our data suggest that the presence of an intact asta gene may represent an additional virulence determinant in both epec groups. the enteroaggregative e. coli heat-stable enterotoxin encoded by asta gene has been found in enteropathogenic e. coli strains. however, it is not sufficient to simply probe strains with an asta gene probe due to the existence of asta mutants and east variants. in this study, epec isolates were tested for the presence of the asta gene sequence by pcr and sequencing. the asta gene was amplified from strains, typical and atypical. sequence analysis of the pcr products showed that strains, typical and atypical, had an intact asta gene. the remaining strains had mutated asta gene with nucleotide deletions or substitutions in the first codons. east geneasta geneenteropathogenic escherichia coli southern blot analysis indicated that asta is located in eaf plasmid in typical strains, and in plasmids of similar size in atypical strains.
 the enteroaggregative e. coli heat-stable enterotoxin encoded by asta gene has been found in enteropathogenic e. coli strains. however, it is not sufficient to simply probe strains with an asta gene probe due to the existence of asta mutants and east variants. in this study, epec isolates were tested for the presence of the asta gene sequence by pcr and sequencing.  the asta gene was amplified from strains, typical and atypical. sequence analysis of the pcr products showed that strains, typical and atypical, had an intact asta gene. a subgroup of atypical strains had a variant type of the asta gene sequence, with four non-synonymous nucleotide substitutions. the remaining strains had mutated asta gene with nucleotide deletions or substitutions in the first codons. the rt-pcr results showed that the asta gene was transcribed only by the strains carrying either the intact or the variant type of the asta gene sequence. southern blot analysis indicated that asta is located in eaf plasmid in typical strains, and in plasmids of similar size in atypical strains. strains carrying intact asta genes were more frequently found in diarrheic children than in non-diarrheic children.  in conclusion, our data suggest that the presence of an intact asta gene may represent an additional virulence determinant in both epec groups. east geneasta geneenteropathogenic escherichia coli 