Enterococcus Faecalis in our Bay Waters?

Marine Microbial Ecology, Research Lessons 2016

Enterococci are commonly found in the intestines of humans, and are used as an indicator of faecal pollution and gastrointestinal pathogens in water being tested. Swimming in recreational fresh or marine water with a large amount of Enterococci is known to be directly related to the risk of gastrointestinal illness.

Indicator Organism

Indicator

Common matrices to be tested

Enterococci

Fecal pollution and the possible presence of enteric pathogens

– Marine water

– Beach water

– Fresh water

Author(s)

Martha Buendia

Introduction

As a member of the Elementary Teachers Engaged in Authentic Math and Science (ETEAMS), I had the opportunity to work with other teachers in a summer research at a microbiology lab at the university of Corpus Christi, TX, TAMUCC. The purpose of the research was to isolate Enterococcus Faecalis,(E.F.), from samples of water of our bay area, two samples pertaining to Cole Park and two samples pertaining to Ropes Park, both located along Ocean Drive in Corpus Christi, TX. After the bacteria isolation, DNA would be extracted to determine if the E.F. is the result of leaking sewer lines or animal feces. Determining the origins/causes of the existence of E.F. in the sample waters would be an important contribution in the field of science to increase awareness to the city representatives and community to develop and implement a plan to take an action to decrease marine water contamination along these recreational parks.

Methods & Procedures

Some questions that I had during the process of this research were: Why is the research focused in Enterococcus Faecalis (E.F.) bacteria and not other bacteria? Why were Cole Park and Ropes Park were selected as the sites to collect water samples? Would there be any other bacteria that should also be a concern to this summer research? To what extent (in regards of number of E.F. colonies) is the presence of this bacteria in our water bays considered an indicator of health risk? Knowing the causes of E.F. existence in the sampling locations is important for middle school students to understand the various factors that pollute the environment in particular marine water of common sites visited by the citizens in their community.

As in any lab, safety is first, and any individual involved in this research must know, observe, and follow the normal safety procedures required in a microbiology laboratory while preparing, using, and disposing of cultures, reagents, and materials, and while operating sterilization equipment.

The method provides a direct count of bacteria in water based on the development of colonies on the surface of the membrane filter. A water sample is filtered through the membrane which retains the bacteria. Following filtration, the membrane containing the bacteria cells is placed on a selective medium, mEI agar, and incubated for 24 hours at 41 ͦ C. In this method, enterococci are those bacteria which produce colonies with a blue halo after incubation on mEI agar. All colonies with blue halo are recorded as enterococci colonies. Magnification and a small fluorescent lamp are used for counting to give maximum visibility of colonies. As a reference, mEI Agar is a selective culture medium recommended for use in the chromogenic detection and enumeration of enterococci in water by the single-step membrane filtration technique.

After counting the E.F. colonies, each colony will be streaked and transferred individually from the mEI agar onto the brain heart infusion (BHI) media, which will be incubated for 24 hours at 41 ͦ C. The following day, isolated colonies will be transfer into Tryptic Soy Broth, (TSB) broth and vortex. The broth containing the each E.F. colonies will be placed in a incubator and rotator at 41 °C overnight (16-20 hours). Thereafter, a 1:1 ratio of the TSB culture and glycerol will be prepared into small tubes, which will be placed in a freezer set to -80°C. ***The glycerol will allow the bacteria to survive even though being frozen. The final step of the protocol is to extract DNA from the TSB/glycerol mix to assure the existence of Enterococcus Faecalis in the sites where the samples were collected.

For detailed methods and procedure, see the following link: Sampling Methods

Results

Colonies that produce a blue halo may be presumptively identified as enterococci fecalis; however, colonies were not always present in each experiment that we conducted. Furthermore, the nature of the experiment did not allow for much data to be taken, as part of the research was conducted on weekends, limiting the ETEAMS participants to take note of the completed research data.

Discussion

In this research, we were able to obtain the results illustrated in the pictures above. The results pertain to samples collected at Ropes Park , (RP), and Cole Park, (CP). The results of blue halo (first picture above) indicate colonies of Enterococcus faecalis present at Ropes Park, and the results of maroon-burgundy (second picture) also indicate colonies of E.F. The difference in color was the result of using different type of agar. At the beginning of the research, we were using a selective agar (unknown name), because mEI agar was not delivered to the lab on time.

It is important to note that repeating a science experiment is an important step to verify that the results are consistent and not just an accident. A typical experiment must be duplicated at least three times, (more is better). Our experiment was repeated five times to be able to collect sufficient evidence to form a conclusion. Throughout the research, colonies of E.F. were not present always in the agar plates. Furthermore, the colonies were only present in the RP samples or in the CP samples, but not both, and the average number of colony forming unit, (cfu), was two per 100mL of water. According to a university graduate, who was working along with us in the lab, “It is believed that the city has a standard of 145 cfu per 100mL of water sample to be considered a health risk/danger to the community.” (This statement must be verified by calling a representative of the Corpus Christi City Water Department). At other times, there was not presence of colonies at all in the agar plates, which was an indicator that the water in our bays is safe to be used for recreational activities.

As a conclusion, the results of the experiment does not indicate a fecal contamination in the samples of water pertaining to RP and CP. However, the final step, extracting DNA will be conducted at a later time by a graduate student. Extracting DNA from the TSB and glycerol culture will provide further information in determining if the E.F. bacteria is the result of human or animal feces. If the results would have had an indication of a great health risk in the bay waters for our community, the results would be presented to our city representatives. If the E.F. was a result of human feces, the probability of sewage leaking into our bays may exists.

It is important to know the quality of our recreational waters, in particular, because we live in an area with a warm climate. Tourism and citizens in this area may opt to go to refresh themselves in our bays in particular during the summer. They must be aware about the conditions and water quality before making decisions of where they may go. Furthermore, our students must be aware of the factors that pollute our recreational waters. Not only trash is a factor, but also other factors may exist. Students must be aware about the existence of Enterococcus Faecalis bacteria and perhaps other types of bacteria that became a threat to our recreational waters. Therefore, we must be conscious about how can we help our environment to be a better place to live and how to maintain a healthy ecosystem.

Lesson Plan