Ehec Genetic Markers

Detection of stx genes is fundamental to PCR assays for EHEC, and there are easily a few dozen primers that have been developed for stx1 and stx2 genes. However, because there are some homologies between toxins, some primers also detect variant types of stx. A study evaluated 14 stx-specific PCR primer pairs by using several STEC strains known to produce stx1, stx2, and/or various Stx variants.[8] All primers specifically detected stx1 and stx2, but varied in the detection of variant forms. However, other primers amplified variant stx genes that, when coupled with restriction fragment length polymorphism (RFLP) analysis, distinguished variant Stx types. PCR for stx identifies only STEC, hence EHEC detection is more complex and requires testing for other markers. Most EHEC carry the chromosomal locus for enterocyte effacement (LEE) pathogenicity island that encodes several virulence factors, including a type III secretion system that mediates the injection of virulence factors into host cells. Among the LEE-encoded virulence genes are eae and tir that encodes for intimin and the translocated intimin receptor, respectively, which coordinately interact to mediate bacterial attachment to the intestinal epithelial cells that initiates the development of the characteristic attachment/effacing lesions.[2] Although there seems to be a strong correlation between the presence of the eae and

Table 1 Genetic markers used in multiplex PCR assays for detection of EHEC

Target

Genes

Shiga toxin

StXi, SÎX2, SÎX2 variants

Intimin

a-eae, ß-eae, g-eae

Enterohemolysin

ehxA (cluster I and II)

Serine protease

espP

STEC agglutinating adhesin

saa

O157:H7

+ 92 uidA

O157, O111, O113 antigens

rfb

H7 flagellin

fliC

stx2 genes with the ability of EHEC to cause illness,[9] LEE is also present in EPEC and, therefore, PCR using eae-specific primers detect both EPEC and EHEC. Consequently, it is essential that eae and stx primers are used in conjunction to distinguish EPEC, which do not produce Stx. There are several eae alleles (a, p, g, a, etc.),[10] which exhibit significant degree of homogeneity, but possess genetic differences that can be exploited to design allele-specific PCR primers to facilitate the identification of EHEC.[10-12] For instance, the p-intimin allele is found in some EHEC serotypes, such as O26 and O111, while the O157:H7-type strain carries the g-intimin gene.[11]

Other putative EHEC virulence factors are found on a 90-kb EHEC plasmid. One of these is EHEC hemolysin, encoded by the ehxA (ehlyA) gene produced by O157:H7 and other EHEC serotypes. The ehxA gene has been proposed as a detection marker for EHEC; however, its presence in some non-STEC strains and absence in strains causing HUS casts some doubts on its role in pathogenesis. Analysis of 79 STEC strains showed that, while highly conserved, there are two distinct ehxA genetic alleles sharing 98% nucleic acid homology.[13] EHEC serotypes such as O157:H7, O26:H11, O111:H8, and O103:H2 maintain cluster I allele, while cluster II is found in EHEC serotypes such as O113:H21 and O104:H21.

Another putative virulence factor on the EHEC plasmid is the espP gene that encodes for serine protease. Although the espP gene is found in O157:H7 serotype, it is absent in atypical German sorbitol-fermenting O157 strains causing HUS.[7,14] Similarly, the saa gene encoding for the STEC autoagglutinating adhesin is found in many strains that do not have eae and speculated to be an alternate mechanism for bacterial attachment. However, saa is more prevalently found in bovine than human STEC strains,[15] hence the role of these adhesins in EHEC pathogenicity remains to be elucidated.

The uidA (gusA) gene, which encodes for p-glucuron-idase, is expressed by most E. coli. One exception, O157:H7, contains the uidA gene but produces a nonfunctional enzyme because of a frame-shift mutation in the uidA gene.[6] The O157:H7 uidA also carries a +92 T to G base substitution that is highly conserved and found only in strains of O157:H7 group.[6] Although not a virulence factor, the +92 uidA mutation has been found to be a unique marker and useful for developing O157:H7-specific PCR assays.[16]

Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

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