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A one shot multiplex Polymerase Chain Reaction PCR was developed for detecting 6 virulent genes of diarrheagenic E.coli from fish and shrimps. In order to differentiate between 5 categories of diarrheagenic E.coli ,I selected the target genes eae for Enteropathogenic E.coli(EPEC), ipaH for Enteroinvasive E.coli (EIEC) ,stx for Enterohaemorragic E.coli (EHEC), aggR for Enteroaggregative E.coli (EaggEC) , elt for Labile toxin producing Enterotoxigenic E.coli , est for Stable toxin producing Enterotoxigenic E.coli (ETEC).Using our multiplex PCR about 18 clinical isolated strains of E.coli in our Laboratory were successfully categorized and easily analyzed for the presence of virulence plasmids .Stable toxin producing ETEC was found to be prevalent among fish and shrimps as 12 cultures gave positive result for est gene ,that is for stable toxin producing E.coli. Primer pairs used in the assay do not interfere with each other and generated amplification product of147 bp. Then the targeted gene is also amplified in a single PCR reaction in one tube and detected by electrophoresis.
Primers ,PCR, EPEC,EIEC,EHEC,EAggEC and ETEC
Escherichia coli is the most important etiologic agent of diarrhea and represents a major public health problem in developing countries. Identification of diarrheagenic E.coli strains requires that these organisms be differentiated from nonpathogenic members of the normal flora. Sero grouping of O-antigen is not sufficient to identify a strain as diarrheagenic ,because it does not correlate ,in most cases with the presence of virulence factors  .Thus identification of diarrheagenic E.coli strains needs to detect factors that determine the virulence of these organisms. With the advent of PCR, it has become possible to detect pathogenic genes in bacterialisolates, allowing the rapid diagnosis of diarrheagenic E.coli  . This study is attempted to develop a multiplex PCR for five pathogenic strains of E.Coli.
Pathogenic strains of E.Coli:
Enteropathogenic E coli: EPEC do not produce any enterotoxins or cytotoxins in the cells to invade, attach and efface. These bacteria induce a characteristic attaching and effacing lesion in epithelial cells in which microvilli are lost and the underlying cell membrane is raised to form a pedestal which can extend outward for up to 10 μm. The genes encoding for this virulence are encoded on the locus of erythrocyte effacement (LEE), a secretion of a number of proteins including EspA and EspB which subsequently trigger a series of responses in the host cell . EPEC presents with watery diarrhoea which can vary in its severity and duration.
Enteroinvasive E.coli: Enteroinvasive Escherichia coli (EIEC) was first shown in 1971 to cause diarrheal disease in otherwise healthy volunteers. It is known to cause shigellosis-like symptoms in both adults and children. EIEC strains cause a disease that is indistinguishable at the symptomatic level from the dysentery . They actively invade colonic cells and spread laterally to adjacent cells.
Enterohemorragic E.coli: EHEC strains bind tightly to cultured mammalian cells and produce the same type of attachment effacement phenomenon as EPEC Strains .Although EHEC strains cause dysentery similar to that caused by shigella species but they do not invade mucosal cells by the production of a toxin,Vero cytotoxic shiga like toxin The clinical features of EHEC infection are most striking. Initial symptoms are of colicky abdominal pain followed by diarrhoea and, in about half of cases, vomiting
EAggEC that bind to small intestinal cells are not invasive and cause no obvious histological changes in the intestinal cells to which they adhere. They differ from ETEC strains primarily in that they do not adhere uniformly over the surface of the intestinal mucosa but tend to clump in small aggregates. EAggEC strains produce an ST like toxin (EAST) and a haemolysin like toxin (120 kDa in size). The infection presents with watery mucoid diarrhoea with no fever or marked blood.
Enterotoxigenic E.coli: ETEC strains adhere to the small intestinal mucosa through colonization factor (CF) antigen and produce symptoms not by invading the mucosa but by producing toxins that act on mucosal cells to cause diarrhea . These strains produce two types of enterotoxins .A cholera like toxin called heat-liable toxin (LT). A diarrheal toxin called heat-stable toxin (ST) (retain 100°C for 30 min) ST is a family of small toxins which can be divided into two groups .methanol soluble (STa) and methanol insoluble (STb). STa causes increase in cGMP levels in the host cell cytoplasm which leads to fluid loss. cGMP like cAMP is an important signaling molecule in eukaryotic cells and changes in cGMP affect a number of cellular processes including activities if ion pumps.LT-I and STa are encoded on plasmids.
Survival of Escherichia coli in the intestine of fish An outbreak of diarrheal illness caused by ingestion of food contaminated with enterotoxigenic E. coli was described in Japan . The illness was strongly associated with eating tuna paste. Brazilian authors isolated 18 enterotoxigenic strains of E. coli (ETEC) from 3 of 24 samples of fresh fish originating from Brazilian markets; 13 of them produced a thermolabile enterotoxin .The authors explained the presence of toxic strains of E. coli in samples collected from fish (not from water) from one fish market by a longer survival of bacteria on an adequate substrate, i.e. inside the living organism. The isolation of 317 E. coli isolates tested for thermostabile (ST) and thermolabile (LT) toxins has been described in another Brazilian study  only one produced ST and none produced LT toxin. Furthermore, although transfer of antibiotic resistance from a fish pathogen did take place in the environment of fish intestinal content, the likelihood of this being a high-risk route for acquisition of resistance by bacteria that can affect humans is low.
When the DNA isolated from those pure cultures are subjected to multiplex PCR by using different primers respected to different virulent genes of E.coli, it is found that 66 % of the cultures had produced stable toxin and there was a 100% positive result from kaanagarta and 60% positive result in the case of shrimps. From the results, it is opined that mere presence of E.coli in fish should not be considered as biological hazard but the presence of virulence genes in the E.coli should be considered as health hazard.
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