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ISSN(p): 2704-2758; eISSN: 2704-2766

Vol. 39, Issue 1, 2020, pp. 34–43 OPEN ACCESS

The Official Research Journal of Western Mindanao State University

Formerly WMSU Research Journal

Screening for Escherichia coli Contamination in Selected

Beach Sands of Zamboanga City, Philippines

Joel Gerone B. Larupay

1, 2* & Izar U. Laput

3

1Philippine Coconut Authority - Zamboanga Research Center, Zamboanga City

2Department of Biological Sciences, College of Science and Mathematics,

Western Mindanao State University, Zamboanga City 3Research Utilization, Publication and Information Dissemination, 2

nd Floor Research Building,

Western Mindanao State University, Zamboanga City

*Corresponding author: joelixir@gmail.com

ABSTRACT

Beach lovers find themselves in a tropical paradise when in the Philippines, where fine

sands characterize most of the beaches. However, due to overexploitation (such as toxic

effluents), beaches are currently undergoing forced restoration, like that of the famous Boracay

Island. This present study aims to provide a scientific support on the current status of Zamboanga

City beaches through evaluating the microbial quality of its coastal sands by explicitly focusing

on Escherichia coli contamination on the sand samples taken from Caragasan, Bolong, and La

Vista del Mar beaches. The extraction of the microorganisms from the sand samples was done

using the standard protocol of the Southern California Coastal Water Research Project

(SCCWRP). The MPN value of the fecal coliforms (>1100) in the three sites exceeds the

standard value of recreational waters. Results suggest that policies regarding sanitation on the

public and private beach resorts should be enforced and adequately observed.

Keywords: microbiology, MPN value, Escherichia coli, Zamboanga City

Larupay, J. G. B. & Laput, I. U. (2020). Screening for Escherichia coli Contamination in

Selected Beach Sands of Zamboanga City, Philippines. Ciência, 39(1), 33–43. [Available

online at www.wmsu.edu.ph/research_journal].

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INTRODUCTION

The Philippines, as situated in the tropical region, is sought after for its lovely beaches

that make the country famous. Most people mark the list on spending the holidays in the beaches

with their family or friends; and a significant percentage of this time is being spent on the shore

than the water itself, such as playing beach volleyball, building sandcastles, and even burying

their bodies under the sand (Ong et al., 2011). However, sand acts as a passive element for

cumulative pollution, which is contaminated by garbage, feces, and water carrying pathogenic

microorganisms and parasites (Sato et al., 2005). These sand microorganisms are attached to the

grains (i.e., not free in interstitial space), which means that they are metabolically active (Nolt-

Helms et al., 2003). Thus, beach sands are an excellent reservoir of infections, such as

contamination with Escherichia coli.

E. coli, a rod-shaped and Gram-negative bacterium, commonly thrives in the lower

intestinal tract of warm-blooded animals, including humans (Lim et al., 2010), and is often

discharged into the environment through wastewater effluent or feces (Jang et al., 2017). It is one

of the fecal indicator bacteria (FIB) that is present in the environment (DOH, 2016). It can cause

various gastrointestinal and respiratory illnesses if it enters body openings or if it contaminates

through open wounds. The US Environmental Protection Agency (USEPA, 2006) emphasized

that swimming in beaches polluted with human waste can cause diarrhea, hepatitis, meningitis,

respiration toxicities and infections of the eye, ear, and skin.

Epidemiological studies conducted between 1953 and 1996 on beaches around the world

suggest a relationship between gastrointestinal symptoms and recreational water quality as

measured by bacterial indicator (E. coli) counts (Halliday & Gast, 2011). According to USEPA

(2006), the standard value for recreational waters (including bathing seawater) is >126 E. coli or

>33 enterococci per 100 ml. Beyond this limit could present a health risk. The World Health

Organization (WHO, 2003) emphasized that sand and other beach sediments should be part of

epidemiological and microbiological studies concerning the ones utilized for recreational

purposes.

In the Philippines, a case of coliform outbreak in 1997 was recorded in the world’s

famous Boracay Island, which was reported by the Department of Environment and Natural

Resources (DENR), and later widely publicized in the national and international media

(Ong et al., 2011). It underwent rehabilitation after more than 20 years (Ignacio, 2018) due to the

uncontrolled growth of fecal indicator bacteria (FIB) (Smith et al., 2011). Environmental

concerns such as high coliform level was raised, which was on a ―thousands or even millions‖

level before its closure to tourists last April 2018 (Endo, 2018). This scientific evidence-based

data provides the basis for the Boracay’s restoration from a cesspool to fortified paradise (Canoy

et al., 2020).

Beach sand has been associated as a source of fecal indicator bacteria (FIB) to coastal

marine waters (DOH, 2016; Yamahara et al., 2007). As Knee et al. (2008) emphasized, sand

may become contaminated by human or animal feces, wastewater, or polluted seawater; or it

may serve as a natural environmental reservoir for FIB. In some countries, recreational area

quality is mainly concerned with water, but in recent years, some studies dealing with the

microbiological quality of sand have been published (Sato et al., 2005).

Zamboanga City in Zamboanga Peninsula lodged a lot of beaches where possible health

hazards can be avoided by knowing the occurrence of these bacteria on beach sands. Thus, this

study was conceptualized to conduct a microbial analysis on selected beach sands (Caragasan,

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Vista del Mar, and Bolong beach) in Zamboanga City. Caragasan and Bolong beaches are

frequently visited public beaches and were chosen as a representative of the west and east coastal

areas, respectively, while Vista del Mar is a privately-owned resort nearest to the city.

Specifically, this study aimed to detect and compare the presence of E. coli on sand samples on

different stations primarily to generate data on the microbial quality of selected beaches in

Zamboanga City, especially those utilized for recreational purposes.

METHODS

Study Site

Sand samples were collected from most frequently visited beaches of Zamboanga City, to

wit: Caragasan, Vista del Mar, and Bolong beaches. The selected beach areas are known to be of

recreational use. Caragasan beach is the most visited beach on the west coast since it is open to

the public with no entrance fee. It is noted that some surrounding residential houses, cottages,

and public sewages which released their waste to the sea. Vista del Mar does not have public

sewages on the area since the beach is privately owned; however it is still open for the public

with an entrance fee, though it has its own sewage for their private drainage. The Bolong beach

is also noted to have houses, cottages, and some domesticated animals wandering in the sand

area.

Sterilization of Materials

Glasswares were sterilized using an autoclave to maintain a more extended period of

sterility. It was wrapped in clean sheets of paper before sterilizing. Sterilization was done at 15 -

20 lbs—pressure for 30 minutes at 121˚C.

Preparation of Media

Different media intended for E. coli analysis were prepared as follows:

Brilliant Green Lactose Broth (BGLB)

This was utilized for the Presumptive test. It was used to detect the presence of coliform

bacteria such as E. coli. Brilliant Green Lactose Broth tubes were prepared following the

manufacturer’s instruction, which was 40 grams to be dissolved in one liter of distilled water.

Ten milliliters of BGLB were transferred in each tube that contained an inverted Durham tube.

All these tubes were cotton plugged and sterilized in an autoclave at 15 lbs. pressure for 15

minutes.

Eosin Methylene Blue (EMB) Agar

Eosin-methylene blue agar is a differential and selective media for gram-negative

bacteria and mainly was used for E. coli. EMB was prepared by adding 37.5 g in one liter of

distilled water. This was heated with continuous stirring to dissolve the agar. The flask was

covered and sterilized in an autoclave at 15 lbs. pressure for 15 min.

Escherichia coli (EC) Broth

To prepare the EC broth, a mixture of 20 g tryptose, 5 g lactose, 1.5 g Bile salts, 4 g

dipotassium phosphate, 1.5 g monopotassium, and 5 g sodium chloride were dissolved in one

liter of distilled water and sterilized. This was used in the completed test for E. coli.

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Research Procedure

Establishment of Sampling Stations

Three sand samples per beach area were utilized for the study, which was obtained from

three stations determined by the total shore length divided by three. This was done once in a

month for three months after the rainy season, to obtain dry sand samples since based on some

studies, E. coli multiplication is higher during warm temperature (Hardina & Fujioka, 1991) and

microbial contamination increased in dry sands (Sabino et al., 2011). Sampling time was done

every last week of August, November, and December.

Collection of Sand Samples

A 100ml wide mouth vial was aseptically filled with the sand sample by scooping the top

layer (one inch deep) at a one-meter distance away from the water line in each sampling station

of Caragasan, Vista del Mar, and Bolong. Collection near the animal feces should be avoided

(SCCRWP, 2010). Samples were immediately transported to the lab on ice and were processed

within eight hours of collection.

Extraction of Microorganisms on the Sand Samples

Ten grams of sand from the sample were weighed using a triple beam balance and were

transferred to the first bottle using a sterilized spatula. Sixty (60) milliliters of sterile, one mole,

and pH 7 Phosphate Buffered Solution (PBS) were added to the bottle and was covered tightly. It

was vigorously shaken for two minutes. The sands were allowed to settle for approximately 30

seconds. Eluant (sand water) was decanted into a second sterile bottle, ensuring no sand particles

were included. An additional 40mL sterile PBS was added again to the first bottle and was

swirled gently for 10 seconds. Immediately, the eluant was decanted and transferred into the

second sterile bottle (SCCRWP, 2010).

Test for the Presence of Escherichia coli

Coliform Analysis

Multiple Tube Method

1.1 Presumptive Test

Nine brilliant green lactose broths were utilized for this test. Each sample was inoculated

with three tubes of 10 ml (double strength), 1.0 ml, and 0.1 ml (both single strength), all with an

inverted Durham tube. These were covered by cotton plugs and were incubated at 37˚C. The

observation was done after 48 h of incubation. A positive result is characterized by the presence

of gas production on the Durham tube.

1.1.1 Most Probable Number (MPN)

The most probable number (MPN) method was used to determine the number of

coliforms by a statistical estimation. This was done by counting the positive tubes on the

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presumptive test and referring to the MPN table. The MPN number is the most probable number

of coliforms per 100 ml of water.

1.2 Confirmatory Test

Melted EMB agar was transferred to the petri dish enough to cover the bottom, and was

allowed to solidify. A loopful of inoculum from positive presumptive test tubes was inoculated

into the EMB agar plate using the streak plate method. It was inoculated 37˚C and observed after

48 h. The presence of metallic green sheen colonies indicated the presence of E. coli and

signified a positive result of this test.

1.3 Completed Test

EC broth was used for the completed test. Each tube was inoculated by pure culture from

the positive EMB agar with 37˚C for 24 h. Gas production indicated a positive result on this test.

Positive tubes were subjected to gram staining.

RESULTS AND DISCUSSION

The gas formation in the Brilliant Green Lactose broth (BGLB) during the presumptive

test indicated a positive result for the sand sample are seen on Fig. 1.

Figure 1. Gas formation in the Brilliant Green Lactose Broth (BGLB) tubes during

the presumptive test.

The combination of these positive tubes was noted for the determination of the most

probable number (MPN) of the fecal coliforms present in the sands. Table 1 shows the average

Most Probable Number (MPN) of fecal coliforms in the sand samples for three replicates after

the Presumptive test.

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Table 1. Average Most Probable Number (MPN) of fecal coliforms in the sand samples for three

replicates after the Presumptive test.

Sampling Sites

Replicates

No. of Positive tubes

MPN Value 0.1 ml 1 ml 10 ml

Caragasan

1 3 3 3 > 1100

2 3 3 3 > 1100

3 3 3 3 > 1100

Vista del Mar

1 3 3 3 > 1100

2 3 3 3 > 1100

3 3 3 3 > 1100

Bolong

1 3 3 3 > 1100

2 3 3 3 > 1100

3 3 3 3 > 1100

As shown in Table 1, the MPN values are the same in the sand samples collected from

Caragasan, Vista del Mar, and Bolong beaches. The MPN, which is >1100 bacteria per 100ml,

was determined from the combination of three of each 10ml, 1ml, and 0.1ml positive tubes after

the presumptive testing. Further confirmatory test showed a distinct metallic green sheen colony

growth, as shown in Figure 2.

Figure 2. Metallic green sheen colony growth in the EMB agar.

The distinct metallic green sheen colony growth in the EMB agar indicates positive

colonies for E. coli. Completed test positive tubes were characterized by the formation of gas on

the Durham tube in the EC broth (Fig. 3 left). This is then further subjected to microscopic

examination via gram staining. Escherichia coli was distinguished as a gram-negative bacillus

when viewed under the microscope with a magnification of 400x (Fig. 3 right).

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Figure 3. Positive results after the completed test.

Table 2 shows the presence of the bacteria Escherichia coli bacteria on the different sand

samples of the beaches at the end of the microbial analysis and the completed test.

Table 2. Presence of Escherichia coli on sand samples after the microbial analysis.

Sampling

Periods

Sand Sample

Caragasan Vista del Mar Bolong

1 2 3 1 2 3 1 2 3

August

+

+

+

+

+

+

+

+

+

November

+

+

+

+

+

+

+

+

+

December

+

+

+

+

+

+

+

+

+

Legend: + E. coli present; - E. coli absent

Caragasan, Vista del Mar, and Bolong beaches were all positive for the presence of E.

coli on the three stations for three sampling periods. This only suggests that the beaches were

either contaminated by the sewages or by the feces. Fine sands characterized all the three

selected beaches. Desamarais et al. (2003) and Lee et al. (2006) both showed that elevated E.

coli thrives in sands with fine grains. Their results were similarly observed in this study that most

of the beach sands of Caragasan, Vista del Mar, and Bolong were a fine sand grain type, and less

of the areas were on medium grain and coarse grain type. This type of sand encouraged people to

choose these beaches.

According to the World Health Organization (WHO, 2003), fecal index organisms can be

used to indicate the degree of fecal contamination. The Mendes’ proposed guideline value of

10,000/10g for sands indicates possible pollution from sewage (Mendes et al., 1993), falls on the

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obtained MPN range of this study. On the other hand, The MPN value exceeds the standard

value of recreational waters (including bathing seawater), which is >126 E. coli or >33

enterococci per 100 ml. This result presents a health risk according to USEPA (2006). However,

although the World Health Organization (2003) considered sand contamination, it made no

recommendations as to standards; neither do they feature in current European or US legislation.

Further tests (confirmatory and completed) showed the presence of E. coli. The three

stations of each selected beach (Caragasan, Vista del Mar, and Bolong) are all contaminated by

E. coli. Four factors were observed and noted to be influential in the presence of E. coli. These

are: the presence of sewage systems, the presence of houses, and some domesticated animals

(like dogs and chickens), the number of bathers, and a generally fine sand grain characteristic of

the beach areas.

Station 3 of Caragasan includes a large sewage system. Accordingly, E. coli is used as an

indicator of sewage contamination, and its possible sources include sewage overflows and

leaking septic systems. Moreover, higher bacterial counts and longer survival time of these

bacteria are in beach areas close to sewage outlets (Whitman & Nevers, 2003). Though the

station 3 of Vista del Mar also has a sewage outlet, it was not functional and only used as a rain

drainage outlet.

Bolong and Caragasan were found to be inhabited by houses with stores and eatery

(―carinderias‖) that are on the beach area. Domesticated animals like dogs and chickens were

also observed to be roaming free on the sands. According to WHO (2003), the principal

microbial risk to human health encountered on beaches is from direct or indirect contact with

animal feces – notably of dogs. The same findings also believed that local flocks like chickens

would elevate E. coli concentration in the area (Whitman & Nevers, 2003). Halliday and Gast

(2011) stressed that sands and sediments offer a habitation to fecal bacterial populations. This is

supported by the American Chemical Society (2007), which states that there are scientific

evidences implicating the beach sand as a reservoir for E. coli. Additionally, the households

within the beach areas are also seen as major causes for E. coli. One mode of fecal pollution is

through the release of untreated human fecal wastes from the nearby septic tanks and canals of

these houses. The WHO (2010) briefly discussed it posing a greater health risk as human wastes

may contain a wide range of pathogens than those from animals.

Fine sands characterized all three selected beaches. Desamarais et al. (2003) and Lee et

al. (2006) both revealed that elevated E. coli thrives in sands with fine grains. Their results were

similarly observed in this study; most of the beach sands of Caragasan, Vista del Mar, and

Bolong were a fine-sand grain type, and less of the areas were on medium-grain and coarse-grain

type. This type of sand encouraged people to choose these beaches. The World Health

Organization (2003) supported this observation by its statement that the number of bathers also

influenced the degree of contamination.

CONCLUSION AND RECOMMENDATION

The study revealed that the sand samples taken in the different stations in Caragasan,

Vista del Mar, and Bolong beaches were either contaminated with feces and sewage fostering

bacterial growth of Escherichia coli. This condition was influenced by the presence of a large

sewage system, the presence of houses and some domesticated animals (like dogs and chickens),

and a generally fine-sand grain characteristic of the beach areas. Policies regarding sanitation on

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the public and private beach resorts should be enforced and properly observed by the

management, nearby residents and the bathers.

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