Identification of Shiga toxin producing E . Coli from raw Meat

Emergence of Shiga toxin genes in Shiga toxin producing E. coli (STEC), all over the world, has become problematic and causes GIT illnesses in human originating in food of animals mainly from poultry. The aim of this study was to render rapid diagnostics of Shiga toxin producing E. coli (STEC) from raw meat. During a 4 month period from May to August a total of 200 samples were collected from beef (100) and chicken (100) and analyzed for Shiga toxin producing E. coli. Of the total of 200 samples of raw meat only chicken (2%) and beef (1%) were detected as PCR positive. Over the past decade many improvements have been made both in conventional and modern technique for detection of bacterial pathogens in food that include sample preparation, plating techniques, counting and identification kits but polymerase chain reaction technique is increasingly used which is considered more specific, sensitive, rapid and cost effective. Raw meat could be a source of Shiga toxin producing E. coli which indicates that possible risks of infections to people could be transferred by the consumption of raw meat and their rapid diagnostics could be made possible by the use of rapid diagnostic technique polymerase chain reaction.


Introduction
In the intestinal micro flora of humans and mammals Escherichia coli (E.coli) are predominantly found.A German pediatrician 'Theodor Escherich' in 1885 discovered E. coli belonging to a family Enterobacteriaceae and this bacterium is one of the inhabitants of intestine.These commensals never cause a problem in their host but some pathogenic E. coli are involved in diarrhea and other enteric illnesses and are called diarrheagenic E. coli [2].These pathotypes indicate the plasticity of E. coli genome and the genome of STEC O157:H7 contains 5416 genes in 5.5x10 6 base pairs of DNA [3] which is considered the most prominent and notorious STEC and its incidence varies according to age group and in most of the cases the etiological agent is food stuff [4].Shiga toxin producing E. coli (STEC) is responsible for causing an increasing number of human outbreaks, characterized by bloody diarrhea, non-bloody diarrhea, hemorrhagic colitis and hemolytic uremic syndrome (HUS) and 85% of these cases are implicated with foodborne transmission.stx is further subdivided into two families' stx1 and stx2 on the basis of sequence analysis.(21).Second and smaller outbreak was identified of different but rare strains of STEC O26 in three different states.A total of 3 states were counted from 3 states that included deaths (0) and hospitalization (1).The number of ill people reported from each state was: Kansas (1), North Dakota (1) and Oklahoma (3) Due to number of constraints, the conventional approaches for detection of foodborne pathogens were replaced by polymerase chain reaction in advanced countries.In Pakistan, however, the PCR has not been currently evaluated for its efficacy for the detection of foodborne pathogens from beef, mutton, chicken and milk due to the paucity of information on the PCR based detection of foodborne pathogens.The conventional approaches to detect food related bacteria rely on the selective enrichment and culture characteristics followed by biochemical characterization.These methods are time consuming, labor intensive and often not reliable in contrast to PCR which is a rapid molecular test with high sensitivity and specificity Material and method A total of 200 samples including raw chicken (100) and beef (100) were collected in PBS from various regions of Lahore and were carried to the laboratory under refrigerated conditions.After the enrichment of these samples in tryptic soya broth, were streaked on MacConkey agar for the identification of E. coli and then on Sorbitol MacConkey agar for the differentiation of pathogenic and non-pathogenic E. coli after overnight incubation at 37°C aerobically.The confirmation of E. coli was done by conducting two biochemical tests, the catalase test using a drop of 3% hydrogen peroxide and gently mixed with the colonies of E. coli isolated of plates and a triple sugar iron test by preparing a butt to the 3/4 th portion of the test tube and then gently pulled back the loop and streaked on the slant area without lifting up the loop.The tubes were plugged properly with cotton plug and incubated at 37°C for overnight aerobically.Finally, the detection of STEC (Stx1 and Stx2) was confirmed by employing a molecular approach polymerase Chain Reaction which proved to be a rapid and reliable method for the detection of foodborne bacterial pathogens.For the detection of Shiga toxin1 (stx1) and Shiga toxin2 (stx2) on all the isolates of E. coli from the samples of raw meat, a molecular approach was conducted i.e. polymerase chain reaction (PCR).This molecular approach was designed for the detection of virulence genes of E. coli isolates.Primers with unique sequences were used for the detection of the targeted genes and a primer sequence is available in tabular form in (Table#1).All PCR reagents were supplied by Thermo scientific USA and all PCR assays were performed in PCR Master Cycler (Eppendorfs Germany).

Primer name
Sequence 5-3 Amplicon size KS7 and KS8 primers were used for the identification of stx1 gene.Final mixture of PCR reaction was 25 μl with 2.0 μl bacterial cells as template, 15.3 μl autoclaved water, 2.5 μl of 10X Taq buffer, MgCl2 1.5 μl, 0.5 μl of dNTPs (5 mM each of dATP, dCTP, dGTP, dTTP), 1.5 μl each of forward and reverse primer (20 pmol) and 0.2 μl of DNA Taq polymerase (5.0 U/ μl).Thermal cycler conditions used for Stx1 were, Initial denaturation was adjusted at 95°C for 10 min.stx1 gene was amplified for 30 cycles, each cycle with 30 seconds of denaturation at 94°C, 1 min for primer annealing at 52°C and 1min for extension at 72°C followed by final extension at 72°C for 5 min and for Stx2 using primers LP43 and LP44, After initial denaturation at 95°C for 10 minutes amplification of DNA product for each cycle were for repeated 30 cycles with denaturation at 94°C for 30 seconds, primer annealing at 57°C for 1 minute, extension at 72°C for 1.5 minutes followed by final extension at 72°C for 5 minutes.

Results
Of the total 200 samples of raw chicken and beef, cultured on MacConkey agar, showed 78 and 65 lactose fermenting pink colonies of E. coli respectively.These colonies were then incorporated for further confirmation of E. coli using biochemical tests.
Triple sugar iron test (TSI) was performed on these pink colonies of E. coli for the confirmation of E. coli in which yellow color of the medium in all test tubes was observed which indicated the fermentation of all three sugars such as sucrose, lactose and glucose that leaded to the determination of E. coli.In addition to TSI, another biochemical test, catalase test was conducted on the same number of pink colonies for the confirmation of E. coli and a moderate reaction was observed with precipitation or bubbling which indicated the presence of E. coli.
After these biochemical confirmations these rose pink colonies of E .coli were further preceded for the determination of STEC using molecular methods.All plates with pink colonies of chicken (78%) and beef (65%) were streaked on SMAC.SMAC displayed Sorbitol fermenting (SF) rose pink colonies 53 and 33 respectively and non-Sorbitol fermenting (NSF) colorless colonies 25 and 32 respectively (Table 2).These NSF colonies of chicken and beef were detected as PCR positive with 2% of chicken and 1% of beef harboring STEC genes either stx1 or stx2 while none of the SF colonies were detected as PCR positive (Figure 1, 2, 3 & 4).

Table 2 . Isolation frequency of E. coli (STEC) from various sources Sampling
sample +ve for E. coli +ve for E. coli +ve for E. coli +ve for E. coli PCR To overcome the PCR inhibition problems and to increase its sensitivity, enrichment method was employed.Enrichment method increased the number of required viable count while dead organisms reduces the probability of detection and even 2µl of the enrichment was able to produce the PCR results which indicate the PCR sensitivity and specificity.ConclusionIn conclusion, I developed a rapid, simple and convenient PCR-based method for the specific detection of one of the major food borne pathogens.This method rendered final results in hours rather than lengthy and equally expensive biochemical methods.