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From 2012.igem.org

Escherichia Coli Case Studies

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Timeline of an outbreak

In order to implement the biosensor correctly, it is imperative to find data about how E.coli outbreaks progressed. Last year, there was on outbreak of E.coli O104:H4 in Germany. It was first detected in early May of 2011, by causing an increased frequency of hemolytic uremic syndrome (Rohde). As the E.coli outbreak continued to spread, traces of E.coli were present in different foods. According to Dr. Rohde in the Open-Source Genomic Analysis of Shiga-Toxin-Producing E.coli O104:H4 case study, the genomic events began with how the E.coli phenotype was determined (Rohde). It wasn’t until five days after the first detection of the E.coli did lab work begin (Rohde). Although this case study reference is a food E.coli outbreak, it continued to grow in size as more food was found to be contaminated. This scenario is similar to a water based E.coli outbreak because typically in larger outbreaks it will affect more than one geographic area. Using this timeline as an example, between the first detection of the outbreak to the E.coli phenotype being determined, it was a period of three weeks. According to research, detection of the pathogen took a very small amount of time compared to determining the phenotype of the pathogen (Rohde). At this point in time there would have been many sick patients and potentially deaths depending on the severity of the outbreak. With our biosensor, this time could be dramatically reduced. Our biosensor could detect ideally any pathogen of interest. In a hypothetical situation, a potential timeline would be that the water would be tested the day a potential contamination would occur. This contamination could come from heavy rain, flooding, development of a new community, natural disaster, etc. Within minutes of the test being initiated, a pathogen would be able to be detected. This would greatly decrease the amount of locals that could get infected with the pathogen because a contamination warning would be implemented the same day. According to current practices and timelines, a water contamination warning would not be able to go out until two days after the pathogen was detected (Pitkanen). Our biosensor would reduce this time by turning days into minutes. By using the team’s biosensor, we are able to engineer our biosensor to a specific phenotype of pathogen. We would be able to do this onsite or have our biosensor pre-designed before its implemented. This would eliminate expensive lab experiments and time by already having the phenotype of the pathogen determined.

Finding the source of an outbreak?

According to the Center of Disease Control and Prevention, a waterborne outbreak is a cluster of two or more infections caused by the same agent(s) and linked to the same water exposure (Centers for Disease Control and Prevention). Outbreaks can be caused by water contaminated with pathogens, chemicals, or toxins which can be spread through ingestion of, contact with, or breathing contaminated water (Centers for Disease Control and Prevention). Though many of these infections occur in developing countries with lower levels of sanitation and less public health awareness, outbreaks have occurred in developed countries such as the United States and Canada (Shannon).For example, in Denmark there was an outbreak of Campylobacter jejuni that happened between the years 1995-1996 (Engberg). This bacterium is similar to E.coli in that it behaves very similar. Campylobacter jejuni is the most commonly reported bacterial cause of diarrhea in humans in developing countries (Engberg). In December of 1995, a procedure was performed to test the amount of nitrate in the ground water. In the process of testing this, a sewage pipe was damaged and then leaked into the ground water supply. It took over a month for the water pump to be turned off from the initial point of contamination (Engberg). For this particular case, a very high coliform count was found in the infected water. “A coliform count is a water contamination test, counting colonies of coliform-bacteria Escherichia coli (E. coli) per 100 milliliter of water. The counted result is expressed as ‘Coliform Microbial Density’ and indicates the extent of fecal matter present (The Law Dictionary). The Environmental Protection Agency, instituted regulations for beaches and recreational waters to ensure that people who came into contact with bodies of water would not become ill. These regulations stated that for freshwater the present single-sample advisory limits are 235 CFU/100 ml for E. coli (Kinzelman). “According to common water quality standards, recreational (fishing and boating) water can have no more than 1000 colonies. Drinking water must be completely free from any colonies, bathing and swimming pool water can have no more than 200 colonies” (The Law Dictionary). In Canada, before water can be re-used, it must go through treatments to eliminate pathogens or particles. It is under normal operating conditions, the efficiency of pathogen removal is measured in the final effluent. This accounts for the amount of wastewater being treated for the removal of pathogens. Under the guidelines issued by Environment Canada, fecal coliforms in final effluents are limited to 400 CFU per 100 mL after disinfection (Shannon). It is at these concentrations which can be harmful to human populations. With the use of our biosensor we would be able to test any water sources to see if pathogens were present. Our biosensor is both portable and efficient, which would allow for finding the source of an outbreak much easier.

What if the E.coli strain mutated or the E.coli was undetectable?

What are the chances of a mutation occurring or what should be done if a mutation occurs?

Currently there is a controversial hypothesis that although E.coli can be detected it is viable but its non-culturable (Bogosian). This topic pertains to our biosensor because there could be pathogens present in water, but not enough to cause an outbreak. This holds true for any bacteria that cannot be cultured by standard methods. A culturable bacterium is inoculated into a sterile microcosm, most commonly seawater or river water, and incubated for a number of days with regular monitoring (Bogosian). This would affect the timeline of an outbreak as well as infect more people with contaminated water. This poses as an obvious public health issue for developing nations who do not have the equipment or technology for this kind of situation. Thus far it has been shown that although this poses as a health risk, it has only been attributed to 22 deaths and 104 total cases in the United States in the last 70 years (Bogosian). It has been shown that although the E.coli is present, the cells actually die off based on their living conditions. A study was conducted to determine if changes in cell populations were due to cell death or to the cells developing into the non-culturable state (Bogosian). This proved to be very useful because the study tested what kinds of environment the E.coli cells would either decline in their populations or thrive to cause an outbreak. The conclusion the E.coli cells did not thrive in non-sterile environments (Bogosian). This proves that when an E.coli outbreak occurs it will be because the cells are in an optimum environment where they are able to grow readily. Although E.coli can be present in water does not mean an outbreak will occur. Our biosensor would ideally be able to detect these pathogens no matter how many colonies were present. Although an outbreak may not occur, the water could still be monitored more closely to prevent an outbreak.

How many tests should be done for an outbreak?

In each of the case studies that were researched, it became apparent that each outbreak has its own characteristics. For example, in 2011, there was an outbreak that took place in Germany where there was a very high incidence in adults, especially women (Rohde). Since each outbreak is unique in its own way, it is difficult to determine how many tests should be done to determine the severity of the outbreak. According to case study about an outbreak that took place in Alpine, Wyoming, physicians began seeing more patients with bloody diarrhea (Olsen). More cases began showing up not only in Wyoming, but also Utah and Washington (Olsen). During the lab investigation of this outbreak, serum was tested from town residents to check for IgM antibodies to the O157 lipopolysaccharides (Olsen). Prior to the outbreak, the Alpine municipal water system was tested multiple times each month for coliform counts. In this case, they were positive results for E.coli in April, May, and June of that year (Olsen). It was in late June that the outbreak occurred, and by middle of July they had found the source of this occurrence. Once the outbreak was detected, stool samples from infected patients were then sent to a lab for further testing. From the time that the outbreak began to the time that the source was found was about 3-4 weeks (Olsen). During this time, tests were being done by multiple agencies such as the Wyoming State laboratory and the Utah Department of Health State Laboratory (Olsen). Using the biosensor that our team has developed, the severity of the outbreak would be extremely decreased. Although regular water samples are taken, once the water tested positive for pathogens, water contamination warnings should have been implemented in ensure that an outbreak was minimalized. With the biosensor designed by the ASU iGEM team, our biosensor would be pathogen specific to detect whether that pathogen is present or not in water. Our biosensor would be able detect pathogens in water within minutes of running the experiment. This would greatly decrease the amount of people that potentially could get sick.

What are the current practices?

E.coli outbreaks have been known to occur in different sources, such as food, water, and plants. It has been shown that before an outbreak takes place in water, there are typically signs that E.coli is present (Olsen). This is because before an outbreak can occur the cells must be able to survive in their environment. In recent case studies it has been found that although E.coli was found to be present in the water supply, although the amount of E.coli was insufficient. It was not until after an outbreak occurred and patients began having symptoms did the amount of E.coli becomes a problem. Once the E.coli outbreak has occurred, it will usually take a few weeks to run tests. These experiments can be determining the phenotype of E.coli or just to determine how many people could potentially be affected by the outbreak. It is quite common during E.coli water outbreaks for the cohort case studies to take place. These studies determine the statistical analysis of the outbreak, where the analysts can see how the outbreak progressed with how many people it infected (Olsen). When E.coli is present in other sources such as food and plants, it is usually not known that this is the source until after the outbreak has already occurred. The biosensor developed by the ASU team, eliminated many of these issues. By engineering the biosensor to already be pathogen specific, it decreases the amount of time to determine what the pathogen. The efficiency of our biosensor allows for the efforts the community to then be focused on water purification, instead of waiting for lab results.

How does the biosensor improve sanitation?

Why is there a need for a real time/faster response for the biosensor needed?

Improving sanitation and hygiene of water has been one of the primary goals for Arizona State’s iGEM team. As a result of producing a biosensor to detect pathogens, it has the ability to not only prevent outbreaks from occurring but also to minimize them. Our biosensor has the ability to be used to detect different kinds of pathogens. This is very useful because if an outbreak were to occur, the amount of time in spending on determining the phenotype of the pathogen can then be spent on eliminating it from water sources. This is another reason why our biosensor can be so effective by being able to detect specific pathogens, but it is also able to give you the answers in real time. This eliminates even more time in the process of determining the pathogen. By being able to prevent an outbreak from occurring, we would be able to improve the sanitation of water, especially in developing countries. It is in developing nations where the outbreaks are the most prevalent. Our biosensor was designed to be easy to use and efficient. This would allow for it to be portable to test many different kinds of water sources. The more our biosensor is used, the more safety and sanitation would be improved.