Escherichia coli Genome sequencing and assembly

Shiga toxin-producing E. coli (STEC) are considered important food-borne pathogens of zoonotic importance causing a variety of clinical syndromes including diarrhea, hemorrhagic colitis (HC), hemolytic uremic syndrome (HUS) and death in humans. STEC associated cases have been reported from all over the world including Pakistan (Bettelheim, 2007; Mohsin et al., 2010). STEC possess a number of virulence genes and most important of these are Shiga toxin (stx) (Louise et al., 1995; Sandvig et al., 1994), E. coli attaching and effacing (eae) and enterohaemolysin (ehxA) (Gyles, 2007; Paton et al., 1997). Ruminants especially cattle and sheep are considered important reservoirs of STEC (Hancock et al., 1998). Humans can acquire STEC infection by direct contact with infected animals or indirectly through contaminated food (EFSA Panel on Biological Hazards, 2013). It is also an important economic concern as the USA has a zero tolerance policy for STEC O157, O26, O45, O103, O111, O121 and O145 (Food safety and Inspection Service, 2011). Only limited information is available about the occurrence of STEC in animals and humans in Pakistan. Therefore, it is of great importance from public health perspective to determine the presence of various STEC in ruminants especially when no previous information is available and compare them with isolates from human STEC cases.The core objective of the proposed study was to find the evidence of occurrence of STEC in slaughtered cattle, buffalo, sheep and goats in Pakistan, if any, and to identify their zoonotic potential.Rectoanal mucosal swab samples were collected from above animal species, slaughtered in various slaughter houses of Pakistan. The samples were enriched in buffered peptone water (BPW) at 37 °C for 24 hours. DNA was extracted and analysed for presence of virulence genes (stx1, stx2, eae and ehxA) using multiplex PCR (Paton and Paton, 1998; Sharma and Dean-Nystrom, 2003). The samples positive for one or more virulence genes were subjected to isolation using Sorbitol MacConkey agar (SMAC). Ten isolates from each plate were analysed for presence of virulence genes (stx1, stx2, eae and ehxA) using multiplex PCR. The isolates were sequenced using Illumina platform to assess their zoonotic potential.