Coliphages as Fecal Pollution Indicators of the Guavate River Basin, Puerto Rico

Leyda Villagrasa, John L. Rivera, RISE Program, UPR Cayey

Abstract:

Coliphages are viruses that infect the bacteria Escherichia coli (E. coli). Coliphages are used as fecal and pathogenic microorganism indicators. They can be found in areas where E. coli thrives, such as water, meat, intestines and feces. E. coli is the most abundant infecting organism in the family of gram-negative bacteria known as Enterobacteriaceae. The purpose of this investigation is to determine if different areas with varying factors affect the quantity coliphages. The varying factors were direct sunlight exposure, high water flow, and low water flow. In this way, it was calculated which area had the most fecal pollution. These were compared with the control sample. Once the samples were taken, the Phages Method was performed due to its advantages such as being cost effective, simple, fast and precise. The results revealed that different factors do affect the amount of coliphages in water. Also, the area with high light exposure demonstrated the most amounts of coliphages indicating the highest fecal pollution. For future works, these samples will be compared with samples of other rivers in urban areas of Puerto Rico.

Introduction:

Bacteriophages are viruses that infect bacteria and that depend on the bacteria to replicate. Their structure is composed of a protein capsid which holds its genetic material and a tail used to inject the genetic material into the host bacteria (Hatfull et al. 2006). Coliphages are bacteriophages that only infect E. coli. Coliphages are generally more resistant than coliforms to chlorination and may have some advantage over coliforms as an indicator of treatment efficiency in disinfected waters. Coliforms are bacteria used as fecal pollution indicators and an example is E.coli (Meloni 2003).

A study demonstrated that visible light decreases the amount of coliphages in water because it affects the metabolic activities of E.coli cells. Some of these activities can be the transport of amino

acids. It also demonstrated prolonged survival of pure E. coli cultures in a warm freshwater pond in temperatures 25°C or a little higher. Low temperature (4ºC) causes a considerable decrease of the survival time of E. coli. In other words, temperatures from around 5 ºC to 35 ºC are the best conditions for the survival of E.coli (Wcislo and Chrost 2000). E. coli also thrives in waters with a pH ranging from 6-8; this is because their inactivation rate stays low within this range (Feng et al. 2003).

Other factors such as dissolved oxygen, conductivity and turbidity affect the amount of E.coli in water. Conductivity is the measure of the water's ability to conduct an electric current. Conductivity depends on the number of ions or charged particles in the water. The unit of measurement for conductivity is expressed in either microSiemens (uS/cm), which is the reciprocal of the unit of resistance, the ohm.

Dissolved oxygen is the amount of gaseous oxygen dissolved in the water. Oxygen enters the water by direct absorption from the atmosphere, by rapid movement, or as a waste product of plant photosynthesis (EPA 2012). Turbidity is the measure of the degree to which water loses its transparency due to the presence of suspended particles (MPCA 2008).

The amount of coliphages that can be found are directly correlated to the amount of E. coli. If conditions are good for E. coli then coliphages most likely be present. Coliphages are important because they are used as E. coli indicators. E. coli is used to evaluate Salmonella in fresh water systems. Every year over 780 million people don’t have access to uncontaminated and drinkable water. Of these, about 3.4 million die from fresh water pollution due to Salmonella, an infectious bacterium that causes many symptoms including diarrhea and typhoid fever, which causes over 200,000 deaths a year (Newton and Mintz 2013). In relation to all of this, it was hypothesized that the varying factors of each area will affect the quantity of coliphages that can be found.

Materials and Methods:

For this investigation, four 500 mL tubes were used to gather water samples. The water samples were taken from four different areas from The Guavate River Basin. Each area had different factors such as direct sunlight exposure, high water flow, and low water flow. In addition, another area was the control group. The control sample was taken between two rocks in an area with water flowing at a normal rate.

After gathering the samples, they were inserted on ice at 4°C. Each sample was divided into three groups (A, B, C) and each group contained four plates, each

plate was labelled according to its group and sample. After aseptically cleaning the work area, 2 mL of E.coli was mixed with 50 mL of water sample and 50 mL of Tryptic Soy Broth (TSB). This was poured into the four plates of each group. This was done for each water sample. The plates were left to solidify for 30 minutes and then they were placed at the incubator at 37°C for 18 hours.

After the 18 hours of incubation, the viral plaques of each plate were counted. Then, the plates were placed in the incubator again for 6 more hours. Once the 24 hours passed, the viral plaques were counted again. The viral plaques for each sample were calculated to find out the amount of plaque forming units (PFU) per 50 mL of water sample. PFU is the amount of coliphages per water sample.

Results:

Figure 1 | Areas where samples were taken

Figure 1 shows the four areas from which the samples were taken. The area highlighted with a red outline demonstrates the where the high light exposure sample was taken,this was sample with the greates amount of PFU’s per 50 mL.

After counting the amount of PFUs

per sample, the data was recorded and showed that the varying factors of each area do in fact affect the amount of coliphages that can be found.

Figure 2 | Table of PFU’s per water sample

As shown in figure 2, the sample with the most amounts of coliphages was the one of high light exposure with 34.7 PFU’s per 50 mL, which is very high compared to the control sample with only 1.67 PFU’s per 5o mL. On the other hand, both the fast flowing water and the stagnant water provided the same amount of .67 PFU’s per 50mL, which is less than the control sample.

Figure 3 | Water parameters from each sample

Water parameters play an important role for the survival of E. coli and coliphages. The water parameters from where the samples were taken showed that the water did in fact have the ideal requirements for the longevity of E. coli and coliphages as seen on figure 3.

Figure 4 | Comparison between a plate from the control sample and a plate from the high light exposure sample

Figure 4 shows how a plate from group C of the control sample had no coliphage plaques, while a plate from group C of the high light exposure sample had four coliphage plaques.

Discussion:

After counting the amount of plaque forming units per 50 mL of sample, the fourth sample shows the highest quantity of coliphages. This demonstrates that the varying factors do affect the quantity of coliphages that can be found. The results of this research show a contradiction with other related studies which state that high light exposure decreases the amount of coliphages that can be found. This could be as a consequence of other factors affecting the fourth sample, since there were a house nearby and green algae growing on the bottom. There was also a sewage pipe from the house very close to the river’s edge, indicating a possibility of fecal pollution and thereby a greater quantity of coliphages.

References:

Feng Y.Y,Ong S.L, Hu J.Y, Tan X.L, Nog W.J. 2003, Effects of pH and temperature on the survival of coliphages MS2 and Qβ, http://link.springer.com/article/10.1007%2Fs10295-003-0080-y

Meloni P1, Isola D, Loi N, Schintu M, Contu A. 2003, Coliphages as Indicators of Fecal Contamination in Sea Water, http://www.ncbi.nlm.nih.gov/pubmed/12838826

Newton A, Mintz E, 2013, Typhoid & Paratyphoid Fever, http://wwwnc.cdc.gov/travel/yellowbook/2014/chapter-3-infectious-diseases-related-to-travel/typhoid-and-paratyphoid-fever

United States Enviornmental Protection Agency, 2012, Dissolved Oxygen and Biochemical Oxygen Demandhttp://water.epa.gov/type/rsl/monitoring/vms52.cfm

Wcsilo R, Chrost R.J. 2000,Survival of Escherichia coli in Freshwater, http://www.google.com.pr/url?sa=t&rct=j&q=&esrc=s&frm=1&source=web&cd=2&ved=0CDAQFjAB&url=http%3A%2F%2Fwww.pjoes.com%2Fpdf%2F9.3%2F215-222.pdf&ei=m1h1U_TqO4iqsQTB-4GICw&usg=AFQjCNFnVeh_3r0MA4JzOABehhtTUW2w4w