a total of 158 stx2-encoding e. coli o157:h7 isolates were assayed , 91 isolates of bovine origin and 67 originally isolated from ill persons ( tables a1 and a2 ) . all isolates demonstrated unique banding patterns on pulsed - field gel electrophoresis ( pfge ) . for polymerase chain reaction ( pcr ) analysis , 5 l of dna obtained from boiled stationary - phase bacteria was added to a 50-l pcr master mix containing a final concentration of 1.5 ( q933 ) or 2.5 ( q21 ) mmol mgcl2 , 200 mol / l each deoxynucleoside triphosphate , 1 u taq polymerase , 0.6 pg/l of primer 595 ( 5-ccgaagaaaaacccagtaacag-3  ) ( 9 ) , and 0.6 pg/l of either primer q933 ( 5-cggaggggattgttgaaggc-3;qstxf ) ( 9 ) or primer q21 ( 5-gaaatcctcaatgcctcgttg-3  ; this study ) . pcr consisted of an initial denaturation at 94c for 5 min ; 30 cycles of 94c for 30 s , 52c ( q933 ) or 55c ( q21 ) for 1 min , and 72c for 1 min ; and a final 10-min extension step at 72c . e. coli strain 933 or fahrp88 was used as a positive control and master mix alone as a negative control . all pcr products were separated by gel electrophoresis ( 100 v ) in 1% agarose gels , stained with ethidium bromide , and visualized by using uv illumination . shiga toxin production was determined by using a commercially available enzyme - linked immunosorbent assay ( elisa ) kit ( premiere ehec , meridian diagnostics , cincinnati , oh ) . briefly , log - phase cells from luria - bertani broth enrichments were diluted to 0.6 optical density ( od ) at 600 nm , subsequently pelleted , resuspended in phosphate - buffered saline , and induced by exposure to uv light ( 240 nm ) for 3 s ( 10 ) . a 1:9 volume of a 10x concentrate of brain heart infusion broth was added to each culture and shaken at 37c for 2.5 h. replicate cultures that were not exposed to uv light ( noninduced controls ) were maintained at 4c . two hundred microliters of each induced and noninduced enrichment was subsequently used as the specimen in the ehec elisa , as described ( 11 ) . od results were recorded for each isolate both with and without uv induction . the relative change in shiga toxin production after induction was calculated for each isolate ; ( odinduced)/odnoninduced ) . e. coli o157 ( edl933 ) and a toxin - negative control isolate were assayed as positive and negative controls each time the assay was repeated . e. coli o157 isolates were classified on the basis of the presence or absence of bands of the predicted size on the q933 - 595 and q21 - 595 pcr reactions ( figure ) . a chi - square test was used to determine whether different pcr genotypes were equally distributed among isolates of bovine and human origin . likewise , a chi - square test was used to assess the equality of distribution of pcr genotypes among bovine isolates from different countries . one - way analysis of variance for nonparametric data ( kruskal - wallis test ) was used to identify differences in ranked - transformed toxin production among noninduced and induced e. coli o157 isolates as well as to determine significant differences in the percent increase in toxin following induction . ethidium bromide  stained gel of the amplification products obtained from q933 - 595 and q21 - 595 polymerase chain reactions . . previously , kim et al . described a nonrandom distribution of e. coli o157 subtypes among cattle and humans by using an octamer - based genome - scanning method ( 7 ) . nine had been previously identified as belonging to the lineage i genotype and seven isolates as belonging to the lineage ii genotype . we found that all nine lineage i isolates consistently amplified the q933 target , regardless of species of origin . all four bovine isolates classified as lineage ii by kim et al . amplified the q21 target . one lineage ii human isolate ( ne015 ) amplified the q933 target , and another lineage ii isolate ( ne037 ) produced no amplicons in either pcr reaction . one human isolate classified as lineage ii ( atcc 43889 ) amplified both target sequences , presumably because of polylysogeny . the distribution of the specific q - gene alleles found upstream of the prophage stx region among bovine isolates may have a geographic component . the distribution of e. coli o157 phage genotypes collected from healthy cattle from diverse geographic areas is consistent with the variable incidences of human disease in different countries ( table 1 ) . for example , six ( 75% ) of eight scottish bovine isolates examined amplified the q933 target , the same target that is frequently present in human isolates of human disease origin . scotland reports some of the highest incidence rates of human e. coli o157related diseases and hemolytic uremic syndrome ( 13 ) . in contrast , none of the seven australian e. coli o157 bovine isolates amplified the 1750-bp fragment . contrary to the situation in scotland and the united states , e. coli o157 infection of humans is rarely reported in australia ( 14 ) . significant difference in proportion of q alleles isolated from different countries ( p < 0.05 , chi - square test for homogeneity ) . the q933 gene target was more commonly identified in human disease  associated strains of e. coli o157 than from strains of bovine origin . amplification of the q933 target , either alone or in combination with amplification of the q21 target from the same isolate , was identified in 60 ( 9% ) of 66 ( 55/66 alone and 5/66 in combination with q21 ; 1 isolate amplified neither target ) compared to 40 ( 44% ) of 91 ( 32/91 alone , and 8/91 in combination with q21 ) of bovine isolates ( p < 0.001 ) . furthermore , these genetic subtypes were nonrandomly distributed among the e. coli o157 isolates of bovine origin obtained from different countries ( p < 0.05 ) ( table 1 ) . these limited data suggest that the distribution of e. coli o157 strains in cattle may differ between countries or regions , thereby providing an explanation for geographic differences in the incidence of human e. coli o157 infection . more isolates from cattle need to be analyzed with these methods to better characterize the e. coli o157 in the bovine reservoir of each country . a positive reaction with the q933 target was significantly associated with higher od results on the shiga toxin elisa ( both noninduced and induced ) and higher - fold increases in toxin production following induction than isolates amplifying the q21 target alone ( p < 0.0001 ) ( table 2 ) . despite these differences , we did not identify any clinical associations between the magnitude of shiga toxin production and severity of human disease could be identified in this study . related virulence factors and host susceptibility are also believed to play essential roles in the outcome of clinical stec infections . the q933-negative isolates obtained from human disease might have lost this q933-containing prophage by the time of isolation , or these isolates might have been recovered from patients also infected with stec containing q933-type prophage ( 15 ) . whether specific q - gene alleles directly correlate with the magnitude of shiga - toxin production or whether other ( unstudied ) factors within the phage lytic cascade genetically linked to specific q alleles instead are responsible for the magnitude of toxin production is not known . the maximum and minimum optical density readings at 600 nm listed in each row are not necessarily from the same isolate ; therefore , the maximum- and minimum - fold increase can not be calculated directly from the table . the antiterminator q , the protein product of the q gene , and pr  , the late promoter , are reputed to be involved in regulating phage late - genes and , because of the location of pr  in prophage genome , of shiga toxin production as well ( 5 ) . in e. coli o157 phage 933w ( genbank no . 15718404 ) , the 359-bp sequence immediately upstream of the stx2 gene is nearly identical ( > 95% nucleotide identity ) . however , further upstream of this area of identity , dna sequences differ significantly . in e. coli o157 933w , this gene is identified as the antiterminator q gene . in contrast , in e. coli o157 stx2vhd this area is occupied by a gene with > 95% sequence identity with the antiterminator q gene of bacteriophage 21 ( gi 4539472 ) . the q gene of bacteriophage 21 does not share dna sequence homology with the q gene of bacteriophage 933w , and only 36% predicted amino acid homology . since the q gene is reputed to play an important role in regulating toxin production , our results provide a plausible explanation ( differential regulation of shiga toxin production ) of why certain e. coli o157 genotypes are more commonly isolated from human patients ( 7 ) .
the antiterminator q gene of bacteriophage 933w ( q933 ) was identified upstream of the stx2 gene in 90% of human disease 
 origin escherichia coli o157:h7 isolates and in 44.5% of bovine isolates . 
 shiga toxin production was higher in q933-positive isolates than q933-negative isolates . 
 this genetic marker may provide a useful molecular tool for epidemiologic studies .