Molecular, Serological And Microbiological Profiling Evidence Of The Formit Transmission Of Escherichia Coli O157: Nm To A 1-Year Old Nigerian Child

Abstract

Transmission of Escherichia Coli O157 by the formit route is under reported since interest in E. Coli O157 is usually arisen during outbreak situations which are mostly linked to consumption of food of bovine origin (Armstrong et al., 1996; Fukushima et al., 1999 and Allison et al., 2000). In December 2005 while working on the isolation of E. Coli O157 from cattle herds in Zaria, Kaduna State, a 1– year old child of a member of the team came down with a mild watery diarrhea. He was not presented to any Physician, since he was observed to be alert, active and with a normal appetite. However, stool specimens were collected from the child on days 2 and 3 of the diarrhea, (which was self limiting on day 3). Stool specimens were also collected from all the five members of his family. All items that the boy had contact with including a laboratory coat, bunch of keys and shoes were swabbed. Finally samples of all the boy's food and drinks were taken. Microbiological, Serological and Polymerase Chain Reaction (PCR) Profiling Assays. l the samples were cultured on Sorbitol - MacConkey (SMAC) agar, after enrichment using Modified Tryptone Soya Broth (MTSB) supplemented with Novobiocin (Oxoid Ltd, Basingstoke, England). All colourless colonies on SMAC agar from the samples were biochemically confirmed as E. coli by the methods described by (Cowan, 1981) and serologically as E. Coli O157:NM using the Remel Wellcolex E. coli O157:H7 kit (Remel Europe Ltd, Kent UK). Shigatoxin profiling was done using the VTEC-RPLA kit (Oxoid Ltd, Basingstoke England). Antimicrobial resistance profiling was carried out by the method of Bauer et al. (1966) using 16 antimicrobial agents: Ampicillin (AMP10), Ceftazidime (CAZ30), Cefuroxime (CXM30), Cephazolin (KZ30), ciprofloxacin (CIP5), compound Sulphonamides (S3300), gentamicin (CN10), Kanamycin (K30), Chloramphenicol (C30), Nalidixic acid (NA30), Neomycin (N10), Norfloxacin (NOR2), streptomycin (S10), Sulphamethoxazole (RL25), Co-trimoxazole (SXT25) and tetracycline (TE30) (Oxoid Ltd, Basingstoke, England). The assays were controlled using a control strain of E. Coli O157:H7 (ATCC 43895). Total DNA was isolated from 1 ml of brain heart infusion (BHI) broth culture grown overnight based on the method of Silhavy et al (1984). The Eae primer (Operon Ltd, Dusseldorf, Germany) was used (Germani et al., 1997). The PCR mix consisted of 2.50μl 10 x buffer, 1.50μl MgCl2, 0.50 dNTPs, eae-f 0.40μl, Taq polymerase (Ampli Taq Gold, Applied Biosystems, California, U.S.A.) 0.50μl, and 4.0μl of template DNA. The volume of the mix was adjusted to 25μl by addition of 15.2μl of nuclease free sterile water. DNA amplification was carried out in a Gene Amp PCR System 9700 Thermocycler (Applied Biosystems, California, U.S.A.) using an initial denaturation step at 94oC for 5 minutes, followed by 30 cycles of amplification with denaturation step at 94oC for 1 minute, annealing at 53oC for 1 minute, and extension at 72oC for 10 minutes and cooled to 4oC.

Keywords: Escherichia Coli O157: NM, Formit, Transmission

Nigerian Veterinary Journal Vol. 28 (3) 2007 pp. 53-55