Food microbiology is an area of study that cover microorganisms that can prevent, create or contaminate food. This area of study also includes the study of microorganisms causing food spoilage. Over the years Escherichia coli O157:H7 has been insulated with increasing frequency from fresh produce. Some produce that are included are bean sprouts, cantaloupes, apples and leaf lettuce. Lettuce is the main focus of this study. E. coli O157:H7 is a strain coming from Enterohemorrhagic Escherichia coli (EHEC), which is a subset of pathogenic E.
coli. This strain can cause diarrhea and or hemorrhagic colitis in human. Hemorrhagic colitis can sporadically advance into hemolytic uremic syndrome. It is also formally known as the Jack in the Box from the outbreak that occurred in Oregon. In this article they examined if E. coli O157:H7 connected with polluted manure or irrigation water can be transferred form the root system into edible portion, putatively by the plant vascular system.
Why is the area of study important?
This area of study important to us as humans because E.
coli it one of the main causes to many diseases within our human digestive system. One of the main ones are food borne, which leads to food poising. With the present of E. coli means that it is a fecal contamination indicator. It is important for use to break down and understand this form of bacteria because it is affecting or daily lives. It is also important because it is said that with proper sanitation methods it prevents the transfer of field found bacteria.
But in this case of E. coli sanitation is not an effective way to prevent E. coli. This strain of E. coli that is being studied in this article is one of the worst ones to come across.
How was each experiment carried out and why?
In their research the authors have five different methods that they performed.
E. coli O157:H7 was converted using the pGFP plasmid, encoding GFP. It was then cultured at 37C for one day and it was cultured in tryptic soy broth supplemented with 100 ug of ampicillin.
The cells were then collected by centrifugation and resuspended in sterile distilled water.
Preparation of planting mixture:
Cow manure collected (48 hrs after evacuation from the animal) and inoculated with a suspension of E. coli O157:H7 and mixed by hand.
The inoculated manure was mixed with soil
The planting mixtures were dispensed into flats, and seeds of green ice lettuce. They kept the flats at 20c, illuminated for 14 hours and was watered daily.
Sampling procedures and detection by culture methods
After day 3, 6 and 9 of planting seeds were collected and they were cut approximant 1 cm from the root system above the soil surface.
The seeds were disinfected on the surface by dipping in 80% ethanol for 5s followed by soaking in 0.1% 〖HgCl〗_2 for 5 to 10 minutes. They were then cleaned two times and allowed to air dry at room temperature.
16 of the 8 seeds that were tested for 5 minutes were placed directly on TSA plates. The left-over seeds were cut and places on TSA- Amp plates.
After incubation for one hour at 37 C the seeds were removed from the plates and incubated overnight.
16 seeds that were in 〖HgCl〗_2 for 10 minutes were examined the same
The plates were illuminated by UV light.
Fluorescence microscopy and laser scanning confocal microscopy (LSCM)
Seed were further tested at days 3,6 and 9. The samples were stained with propidium iodide for 30 minutes, then washed two times in phosphate-buffered saline and then displayed on a glass slide to be observed.
Seeds that had surface- associated E. Coli O157:H7/pGFP were looked into more using LSCM to determine were the target pathogen was located.
Effect of irrigation with contaminated water and Manure slurry
To try to figure out whether straight surface contact with the ripe portion of the plant is necessary for internal contamination 25 green ice lettuce plants were grown.
They fertilized the lettuce plants weekly
The mature plants were relocated and bundled with twine to prevent them from touching the soil. They were then irrigated with containing 〖7.5×10〗^7 CFU of E. coli O157:H7/ pGFP 〖ml〗^(-1).
After contaminating the plants 5 were pulled after 1, 3 days. They sliced the plants 2 cm above the soil and the ripe portion of the placed was merged with 200 ml of SWD and homogenized for 2 minutes.
The liquid portion was isolated, centrifuged, resuspended and assigned on a TSA- Amp plate. Overnight the plates were incubated at 37C and colonies were examined.
What were the general results of the paper?
In this study they found that 10-minute exposure of exterior surfaces of seeds 〖HgCl〗_2 was able to remove most culturable bacteria if the target pathogen was discovered within the seed tissue and was safeguarded from the sanitizing agent. E. coli O157:H7 maintained the plasmid encoding GFP. They also found that the surface of the seeds was most likely to become contaminated as they grew and penetrated the soil surface. E. coli can localize within lettuce tissue and it is capable of inserting the roots of mature lettuce plants and can be carried upward to locations within the ripe portions of the plant.
What were the general conclusions made by the authors for each experiment?
The conclusions that the authors gathered from this research is that one there doesn’t have to be direct contact among leaves and a contamination source is not essential for the organism to become incorporated into ripe lettuce tissue. They also have demonstrated that lettuces grown in soil containing contaminated manure or irrigated with contaminated water results in contamination of the ripe part of the lettuce plant. Furthermore, the results suggest that the lettuce plant doesn’t have to be in direct contact with the pathogen it can become contaminated through the root system. The sanitizing of lettuce is not a validity method to remove all E. coli O157 cells. The isolation of a large number of organisms, as a result of their subsurface position is mostly the reason of the lack of success of surface- sanitizing treatments.
List numerically all of the scientific limitations of the paper?
A limitation that my study faces was that the quantities of E. coli O157:H7 that was used in the study were far more that the amounts that may be found in an agricultural field.