MOLECULAR DIAGNOSTICS OF FOODBORNE PATHOGENS

All over the world Shiga toxin producing E. coli (STEC) are frequently cause of 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 and milk. During a 4 month period from May to August a total of 300 samples were collected from beef (100), chicken (100) and milk (100) and analyzed for Shiga toxin producing E. coli. Of the total of 300 samples of raw meat and milk only chicken (2%), beef (1%) and milk (1%) were detected as PCR positive.Raw meat and milk 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 milk 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. It harmlessly colonizes the intestine but several strains of E. coli cause various intestinal diseases. E. coli are described as Gram negative, facultative anaerobe, non spore forming; rod shaped and are generally motile. [1]. It has been divided into six pathotypes that includes atypical enteropathogenic E. coli (ATEC), enteropathogenic E. coli (EPEC), enteroaggregative E. coli (EAEC), enterotoxigenic E. coli (ETEC), enterohemorrhagic or Shiga toxin producing E. coli (EHEC/STEC) and enteroinvasive E. coli (EIEC) [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, nonbloody diarrhea, hemorrhagic colitis and hemolytic uremic syndrome (HUS) and 85% of these cases are implicated with food borne transmission. stx is further subdivided into two families’ stx1 and stx2 on the basis of sequence analysis. The cardinal virulent factors, of STEC are stx1 and stx2 and are referred as cytotoxic [2, 5]. The association of STEC outbreaks has been considered with a variety of dairy products especially


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.It harmlessly colonizes the intestine but several strains of E. coli cause various intestinal diseases.E. coli are described as Gram negative, facultative anaerobe, non spore forming; rod shaped and are generally motile.[1].It has been divided into six pathotypes that includes atypical enteropathogenic E. coli (ATEC), enteropathogenic E. coli (EPEC), enteroaggregative E. coli (EAEC), enterotoxigenic E. coli (ETEC), enterohemorrhagic or Shiga toxin producing E. coli (EHEC/STEC) and enteroinvasive E. coli (EIEC) [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, nonbloody diarrhea, hemorrhagic colitis and hemolytic uremic syndrome (HUS) and 85% of these cases are implicated with food borne transmission.stx is further subdivided into two families' stx1 and stx2 on the basis of sequence analysis.The cardinal virulent factors, of STEC are stx1 and stx2 and are referred as cytotoxic [2,5].
The association of STEC outbreaks has been considered with a variety of dairy products especially raw meat and milk if they are not properly cooked or boiled [6].A number of reports have been submitted on the sources of transmitting the STEC such as water [7], yogurt [8] and airborne [9] as well.
Human outbreaks of STEC are more derived from ground beef than any other source [6].In particular, ruminants and cattle are considered the primary source of E. coli O157:H7 [10,11].It was also reported that person to person contact is also a source for the transformation of STEC and it was also manifested that STEC is shed by the infected person through stool even after a month of recovery [12].
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 300 samples including raw chicken (100), beef (100) and milk (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 and milk, 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 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 300 samples of raw chicken, beef and milk cultured on MacConkey agar showed 78, 65 and 57 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%), beef (65%) and milk (57%) were streaked on SMAC.SMAC displayed Sorbitol fermenting (SF) rose pink colonies 53, 33 and 28 respectively and non-Sorbitol fermenting (NSF) colorless colonies 25, 32 and 29 respectively.These NSF colonies of chicken, beef and milk were detected as PCR positive with 2% of chicken, 1% of beef and 1% of milk, harboring STEC genes either stx1 or stx2 while none of the SF colonies were detected as PCR positive.

Discussion
Foodborne infections are caused by the consumption of contaminated food with bacteria, parasites and viruses (15).Shiga toxin is a deadly bacterial strain usually causes foodborne illnesses and often associated with beef contamination, giving weekend state to the young, weak or elders.E. coli appears in various forms like O157, producing Shiga toxin along with the emergence of HUS ( 16).
The pathogenicity of E. coli O157:H7 is ascribed with some virulence factors such as Shiga toxins Stx From 300 samples of raw meat and milk E. coli was identified using plate methods incorporating MacConkey agar, Sorbitol MacConkey agar, triple sugar iron test and catalase test.Sorbitol MacConkey agar differentiated NSF and SF.These NSF colonies were further tested for PCR as these colonies were regarded as pathogenic.It is revealed from a study that from 220 samples of diarrheagenic patient only 59% was having E. coli using MacConkey agar as initial identification.
In present study the collection of samples was done between the months June to August and it was noted the positive testing was prevalent during these months.The elevation in ambient temperature is more favorable and plausible for incidence of STEC, thereby resulting in the constant source of infections.
According to this information on elevation of temperature, the prevalence of STEC could be expected more, as observed, in summer season from June to August and this pathogen STEC was found in this duration to the detectable level in raw meat and milk.
In a survey of United Kingdom, it was reported that the positive testing of E. coli O157:H7 was only in the months between May and September.The positive testing of E. coli O157:H7 was more prevalent between June-September in United State (19).
In present study for the identification of STEC a PCR approach was employed using gene specific primers targeting virulent genes Stx1 and Stx2 and monitored with a reference strain EDL933.The specificity of PCR was validated using a reference strain and was further evaluated on 300 isolates of raw meat and milk of different geographical regions.The PCR approach was conducted for rapid and reliable identification of STEC on enrichment of samples In conclusion, a rapid, simple and convenient PCRbased method was developed 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.Application of this method in food ).All PCR reagents were supplied by Fermentas USA and all PCR assays were performed in PCR Master Cycler (Eppendorfs Germany).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

1 and
Stx 2 (17) which are encoded with Stx family and cytotoxic effects are manifested by this pathotype on endothelial cells of kidney, heart and brain (2).
instead of using DNA isolation procedures and plate labor intensive methods to replace the traditional tedious techniques.Out of 300 samples of chicken, beef and milk only 4 (1.3%) number of samples were detected as positive harboring STEC genes either stx1 or stx2.Of the 4 (1.3%), hundred samples of each chicken, beef and milk carried 1% and 1% respectively.Detection methods based on nucleic acid have been developed for nearly all bacterial pathogens, based on hybridization assays PCRs targeting the gene region or specific sequence of a gene.Several problems may be encountered for the direct detection of pathogens from the food samples: contamination in sensitive PCR assay, PCR inhibition by food components and detection of living as well as dead cells (20).As described above two virulent genes were selected from the bacterium Shiga toxin producing E. coli: Stx1 and Stx2.By incorporating the PCR primers and other components, became able to develop a PCR for the detection of these genes.An important factor to evaluate DNA based test is the specificity of DNA sequence chosen for gene of interest which is going to be targeted (Winters et al., 1998).The ability of PCR is to detect or amplify a gene or a segment of a gene directly from the sample containing various bacterial strains.One major problem in detecting the gene directly from the sample which often encountered with PCR is the reduction in sensitivity (21).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.