Occurrence of Shiga toxin producing E . coli from raw milk

Shiga toxin producing E. coli (STEC) causes GIT illnesses in human originating in food of animals all over the world. The aim of this study was to make rapid diagnostics of Shiga toxin producing E. coli (STEC) from raw milk. During a 4 month period from May to August a total of 100 samples were collected milk (100) and analyzed for Shiga toxin producing E. coli. of the total of 100 samples of raw milk only milk (1%) were detected as PCR positive. Raw 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 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, nonspore forming; rod shaped and is generally motile.[ 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].In New Hampshire and Vermont associated with a particular restaurant in eastern Vermont an outbreak of STEC has been detected while the source of outbreak has not been identified.Although one confirmed and two probable cases associated with the outbreak were reported in New Hampshire during a period from August 27, 2015 to September 26, 2015, with illness onset from September 5, 2015 to October 1, 2015 (13).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 100 samples of raw milk 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 nonpathogenic 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).All PCR reagents were supplied by Thermo scientific USA and all PCR assays were performed in PCR Master Cycler (Eppendorfs Germany).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 100 samples of raw milk cultured on MacConkey agar showed 57 lactose fermenting pink colonies of E. coli.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 milk (57%) were streaked on SMAC.SMAC displayed Sorbitol fermenting (SF) rose pink colonies 28 and non-Sorbitol fermenting (NSF) colorless colonies 29 (Table 2).These NSF colonies of milk were detected as PCR positive with 1% of milk, harboring STEC genes either stx1 or stx2 while none of the SF colonies were detected as PCR positive (Figure 1).

Table 2 . Isolation frequency of E. coli (STEC) from various sources Sampling
[20]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[20].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.Application of this method in food industries and municipal water supply departments is an additional benefit attributed to this technique.
sample +ve for E. coli +ve for E. coli +ve for E. coli +ve for E. coli PCR