INT J CURR SCI 2015, 17: E 97-104

RESEARCH ARTICLE ISSN 2250-1770

Bacteriological analysis of piped water and purified drinking water

in Dasmariñas city and Bacoor city (Philippines)
Francisco EE, NP Panganiban and Norbel A Tabo*
Biological Sciences Department, College of Science and Computer Studies, De La Salle University-Dasmariñas,
DBB-B Dasmariñas, 4115 West Avenue Cavite, Philippines
*Corresponding author: natabo@[Link]
Abstract

This study assessed the bacteriological quality of piped water and purified drinking water in Dasmariñas City and

Bacoor City, Cavite. Forty-eight water samples were collected at random from two cities. The water samples were subjected

to heterotrophic plate count (HPC) method. Afterwards, positive results from HPC were transferred to Salmonella-Shigella

Agar (SSA) and Eosin Methylene Blue Agar (EMBA). The isolates from the SSA and EMBA were enriched to nutrient

broth. In Dasmariñas City, 33.3% was observed in purified drinking water, while no sample rendered positive in piped water.

In Bacoor City, both of the samples showed positive results. In purified drinking water, 33.3% was observed, while in piped

water, 58.33% was positive to HPC. No significant difference (p>0.05) was observed between the type of water in relation to

HPC. However, there was a significant difference (p=0.035) between the sample collection site in relation to HPC. The four

isolates were confirmed as S. Typhi (one isolate) and E. coli (three isolates) using analytical profile index (API) 20 E

identification kit. This study concluded that water samples collected in two cities that showed positivity in HPC and presence

of S. Typhi and E. coli are not safe for human consumption.

Keywords: API, E. coli, HPC, S. Typhi

Received: 14th April 2015; Revised: 28thMay; Accepted: 14thJune; © IJCS New Liberty Group 2015

Introduction diarrhea, cholera, and gastroenteritis (WHO, 2004). In

Water is one of the natural resources needed by the Philippines, water is treated in treatment plants and

humans for their survival. It is used for preparation and distributed by water concessionaires. The water source of

consumption of food, washing and bathing. Aside from Bacoor City, supplied by Maynilad uses Putatan

these, it may also serve as a mode of transmission for Treatment Plant to treat raw water from Laguna Lake. It

microbes like Escherichia coli, which produce toxins that has fourteen units of microfiltration assemblies and six

cause gastrointestinal disturbances and Salmonella reverse osmosis assemblies (Maynilad, 2009).

Typhi, which causes typhoid fever (Tortora et al., 2013). On the other hand, the water source of Dasmariñas

If the water used for human consumption does not City supplied by Dasmariñas Water District (DWD)

undergo a well-processed treatment system, this can give treats its water by chlorination after filtration (DWD).

a higher risk of causing diseases to consumers. Thus, The application of chlorine is used to disinfect water

infants and children who have low immune system will supply from possible contamination. This is to ensure

get higher chance of suffering from diseases such as that water being supplied to consumers is safe, clean and
 Francisco et al., 2015

potable. However, water could still be a source of Region IV-A from 2008 to 2009 (DOH, 2009). The child

infection after it has undergone several purification mortality of diarrheas and gastritis of presumed

processes (Demirel et al., 2011). In 2009, there were 685 infectious origin per 100,000 populations in the

mortalities recorded of intestinal infectious disease in Philippines from 2008 to 2009 showed a thirteen percent

Region IV-A, which makes intestinal infectious disease increase for ages 1 to 4, a constant child mortalities for

as one of the leading causes of death in the region having ages 5 to 9 while no mortalities were recorded for ages

a 4% increase compared to its previous year (DOH, 10 to 14 (DOH, 2009). Waterborne diseases threaten the

2009). Aside from diarrhea, studies showed that the human health even after the water has undergone modern

ingestion of contaminated water with coliforms of techniques like disinfection, sanitation and water

Escherichia coli, Salmonella sp, Vibrio cholerae and purification (Momtaz et al., 2013). Assessing the

other microorganisms can be the source of serious microbiological quality of piped water and purified

complications like enteritis, death, economic loss and drinking water is needed in order to monitor the

also malaria, cholera, dysentery and hepatitis A (WHO, occurrence of Escherichia coli and Salmonella Typhi

2004). along with other pathogens that cause waterborne

Acute watery diarrhea (AWD), Typhoid and diseases. This is to measure the number of occurrences of

Paratyphoid Fever are some of the leading causes of diarrhea, dysentery, enteritis and death especially here in

morbidity in the Philippines. There were 434,445 the Philippines wherein our water resources are easily

morbidities recorded in 2008 and 322,799 morbidities exposed to sewage and pesticides due to our agricultural

recorded in 2009 for AWD. This showed a decrease of activities. This study assessed the water quality of

25% percent in total number of morbidities of AWD purified drinking water and piped water in Dasmariñas

from 2008 to 2009. There were 11,119 morbidities City and Bacoor City.

recorded in 2008 and 6,279 morbidities recorded in 2009 Materials and Methods

per 100,000 populations. A decrease of 44% in total This study used a descriptive research design in

number of morbidity per 100,000 populations was from assessing the water quality of purified drinking water and

2008 to 2009 (DOH, 2009). The infant mortality of the piped water in Dasmariñas and Bacoor City.

diarrhea and gastritis of presumed infectious origin per Heterotrophic plate count was used in determining the

1000 live births in the Philippines showed a three percent heterotrophic bacteria present. The selective plating was

increase from 2008 to 2009. There were 937 mortalities done for preliminary assessment while the Analytical

recorded in 2008 and 971 mortalities recorded in 2009 Profile Index (API) kit was used in confirmation of the

(DOH, 2009). The intestinal infectious disease is one of identity of isolates. Forty-eight (48) samples were

the leading causes of death in Region IV-A. There were collected within the limits of Dasmariñas City and

656 mortalities recorded in 2008 and were 685 Bacoor City.

mortalities recorded in 2009. This showed a four (4) Sample collection: In total, forty-eight (48) samples

percent increase of the intestinal infectious disease in were collected within the two cities. The water samples

[Link]
 Francisco et al., 2015

consisted of 12 samples of piped water and 12 samples of obtained by referring to the analytical profile index (API)

purified drinking water were obtained from Dasmariñas (Imen et al., 2012).

and Bacoor, cities of Cavite. The water samples were Statistical analysis

collected randomly in Dasmariñas City and Bacoor City. Colonial characteristics were observed in R2A,

One hundred milliliters (100 mL) of piped water was SSA, and EMBA plates. Such characteristics include

collected and placed in a sterilized bottle. One hundred size, form, margin, surface elevation, consistency and

milliliters (100 mL) of purified drinking water was pigmentation. Analytical Profile Index (API) 20E ID kit

bought from the local stores located within the two cities. was used to identify E. coli and S. Typhi, which uses 20

All water samples were transported to the laboratory miniaturized biochemical tests. Color changes in every

after the collection and processed immediately. test were recorded. Chi square test was used to determine

Heterotrophic plate count the difference between piped water and purified drinking

The bacteriological assessment of water quality water in relation to HPC. It was also used to determine

used the heterotrophic plate count method. Briefly, 1 mL the difference between source water of Dasmariñas and

of the water sample was mixed with liquefied R2A Bacoor City, Cavite. All statistical analysis was

medium. It was allowed to solidify for 5 mins. After 5 conducted using STATA 9.0 for windows (STATA

mins, the plates were incubated at 37oC for 24-48 hrs. Corp., College Station, Texas 77845 USA). Significance

After incubation, the counts were determined and the was defined as a probability of less than 0.5 (p< 0.05).

results were expressed as cfu/mL (colony-forming unit

Results and Discussion
per milliliter) (Zeenat et al., 2009).
The assessment of bacteriological quality of
Preliminary Identification of Escherichia coli and
drinking water for fecal contaminants is very important
Salmonella Typhi
due to increasing diarrheal and typhoidal diseases in the
In the preliminary identification of E. coli and S.
Philippines (CIA US 2014). In Philippines, 322,799
Typhi, selective plating method was used. The colonies

from the HPC positive were streaked in eosin-methylene cases were recorded for acute watery diarrhea in 2009

blue agar for E. coli (EMBA) and Salmonella-Shigella and still one of the leading causes of morbidity (DOH,

Agar for S. Typhi (Imen et al., 2012). 2009). This study was conducted to assess the

Confirmation of the identity microbiological quality of piped- and purified drinking

Isolates were identified using API 20 E water in Cavite province, Philippines. Table 1 shows the

(BioMerieux®) identification kit. Colonies were heterotrophic plate count of 24 piped water samples and

obtained from purified cultures in eosin-methylene blue 24 purified drinking water samples collected from

agar (EMBA) and Salmonella-Shigella Agar (SSA) and Dasmariñas and Bacoor City. In Dasmariñas City, 33.3%

grown in separate tubes containing 10 mL Nutrient broth (4/12) were observed in purified drinking water, while no

inoculated at 370C for 24 hrs. Reactions were read sample rendered positive in piped water. In Bacoor City,

according to the reading table and identification or were both of the type of water samples showed positive

[Link]
 Francisco et al., 2015

results. In purified drinking water, 33.3% (4/12) were Dasmariñas City. The heterotrophic plate count indicates

observed, while in piped water, 58.33% (7/12) were the contact of water with sewage or inadequate treatment

positive to HPC. There are a higher number of or post treatment contamination (Sarwar et al., 2011).

heterotrophic plate counts for both piped water and The purified drinking water collected randomly from

purified drinking water in Bacoor City than in Dasmariñas and Bacoor City undergoes

Table 1. Heterotrophic plate count (HPC) of the water samples

Samples Number samples Number w/ HPC positive

(>500 cfu/mL)

Piped water

Dasmariñas 12 0
Bacoor 12 7

Purified drinking water

Dasmariñas 12 4

Bacoor 12 4

Table 2. The presence of S. Typhi and [Link] in water samples

Samples Presence of S. Typhi Presence of E. coli

Piped water
Dasmariñas 0 0
Bacoor 0 1

Purified drinking water

Dasmariñas 1 0

Bacoor 0 2

distillation and filtration (EPA 2005). In Dasmariñas water samples from both Bacoor and Dasmariñas Cites.

City, out of 12 purified drinking water samples brought This is due to the absence of disinfection treatment after

from local water drinking stations, 4 were HPC positive bottling where an increase in bacterial content had

and in Bacoor City, out of 12 purified drinking water sourced due to the revivable colony count (Demirel et al.

samples brought from the local water drinking stations, 4 2011). The presence of microorganism after the water

were HPC positive. The bacterial growth is higher in treatment is due to biological activity occurring in water

bottled water because plastic is more permeable to treatment equipments wherein the filter media held

oxygen and extraneous vapors and because it also together the bacteria and provides adequate food supply

releases nutrients that increase bacterial growth. for the bacteria (DES 2010). In Dasmariñas City, the

Bacterial adherence to bottle surfaces aid in its survival piped water undergoes filtration and chlorination

process (Al-Qadiri et al., 2010). Purified drinking water (DWD). Our results showed the effectiveness of

samples contain more coliforms than that of the piped chlorination in water treatment process since there is no

[Link]
 Francisco et al., 2015

HPC positive in the 12 piped water samples collected piped water and purified drinking water in relation to

randomly in Dasmariñas City. Chlorination is used by heterotrophic plate count (HPC). This means that the

water processing plants in water treatment. It shows to be water type does not affect the number of HPC. However,

effective for killing bacteria and viruses but not for some there was a significant difference (p=0.035) between the

protozoan cysts (SDWF). While in Bacoor City, the water samples from Bacoor and Dasmariñas City in

piped water undergoes large-scale microfiltration and relation to HPC. This means that the collection site

reverse osmosis (Maynilad). Our results showed that out determines the number of HPC which varies depending

of 12 water samples collected randomly in Bacoor City, on the location where the samples are collected.

seven were HPC positive. The presence of

microorganism after the water treatment is due to

plumbing work occurring and also through the growth of

microorganisms as biofilms on surfaces (e.g. pipelines)

in contact with water (DES, 2010).

Bio-fouling is caused by biologically active

microorganisms bacteria, fungi and other eukaryote

microorganism. It is a dynamic process of microbial

colonization and growth, which results in the formation Table 2 shows the presence of S. Typhi and E. coli

of microbial bio-films. This is the biggest problem in of 24 piped water samples and 24 purified drinking water

membrane filtration because even with highly effective samples collected from Dasmariñas and Bacoor City.

pre-treatment processes that can remove 99.9% of all The colonies from heterotrophic plate count (HPC)

microorganisms, there are still enough cells left which subjected to Salmonella-Shigella Agar and eosin-

can colonize and form a bio-film causing flux decline methylene blue agar were identified and observed. In

and irreversible fouling. This is a result of formation of Salmonella-Shigella agar, presence of black colonies (S.

bio-films on membrane surfaces. Once bacteria attach to Typhi) was only observed in one sample from a purified

the membrane surfaces, they start to multiply and drinking water collected at Dasmariñas City. Wherein

produce extracellular polymeric substances (EPS) to eosin methylene blue agar, presence of metallic green

form a viscous, slimy, hydrated gel (Pandey et al., 2012). colonies (E. coli) were found in both types of water

Thus, water quality in Bacoor City is polluted by fecal samples (one in piped water and two from purified

contaminants, leaking sewage lines, human and animal drinking water) collected from Bacoor City. No metallic

excreta flowing into open drain system, which is the green colonies (E. coli) found from any type of water

common potential source of contamination in defective samples collected from Dasmariñas City. Figures below

drinking water distribution system (Din et al., 2014). No show the S. Typhi and E. coli isolates. Colonies

significant difference (p>0.05) was observed between resembling S. Typhi and E. coli were confirmed

[Link]
 Francisco et al., 2015

Table 3. Confirmation of isolates using analytical profile index

Test code Substrate Isolates

1aS1 2cE2 4cE1 2dE1

ONPG ß-Galactosidase - + + +

ADH Arginine dihyrolase - - - -

LDC Lysine decarboxylase + + + +

ODC Ornithine decarboxylase + + + +

CIT Citrate utilization - - - -

H2S H2S production + - - -

URE Urea hydrolysis - - - -

TDA Deaminase - - - -

IND Indole production - + + +

VP Acetonin production - - - -

GEL Gelatinase - - - -

GLU Glucose + + + +

MAN Mannitol + + + +

INO Inositol - - - -

SOR Sorbitol + + + +

RHA Rhamnose + + + +

SAC Sucrose - + + +

MEL Melibose + + + +

AMY Amygdalin - - - -

ARA Arabinose + + + +

using API Kit. The results showed that the isolates for their identification number. The identification

conform to S. Typhi and E. coli. For confirmation of the number of 1aS1 is 4504552 which is classified as S.

positive results from eosin methylene blue agar and Typhi. The 2cE2, 4cE1 and 2dE1 share the same

Salmonella-Shigella agar, the isolates were subjected to identification number, 5144572 which is classified as E.

BioMerieux® API 20 E ID KIT. Table 3 shows the coli. The presence of fecal indicator bacteria, including

results of 21 tests for each isolate. At each test, the coliforms, S. Typhi and E. coli, indicates the potential

positive and negative colors are noted for assessing the presence of enteric pathogens and related human health

identification of each isolates. The results are tabulated risk, as these microorganisms are commonly found in

[Link]
 Francisco et al., 2015

mammalian intestines (Smith et al., 2014). Although [Link]

most of these organisms do not cause illness to human, ctsheets/dwgb/documents/[Link]

they can cause nuisance through generation of taste and Din M, Ahmad Z, Aleem A, Pirkani GS, Mohammad A,

odor or discoloration of drinking-water supplies Ahmad N (2014). Pathogens From Drinking

(PNSDW 2007). Water; Isolation and antibiogram of pathogenic

Conclusion organisms from drinking water in Quetta city.

Assessing the water used for consumption is very Professional Med J 21(4): 760-765.

important because this water source would determine the EPA (2005). Water Health Series: Bottled Water Basics.

health condition of the consumers. The water quality 816-K-05-003.

according to the Philippine National Standards for [Link]

Drinking Water (PNSDW) states that the water sample s_bottledwater.pdf

should be free from any pathogenic bacteria. Hence the Imen BS, Ridha M, Mahjoub A (2012). Laboratory

samples, positive to HPC and [Link] and [Link], were Typing Methods for Diagnostic of Salmonella

recognized as non-potable. Bacoor City has higher strains, the Old Organism That Continued

bacterial load for both piped water and purified drinking Challenges. Salmonella-A Dangerous Food borne

water than that of Dasmariñas’s. This could mean that a Pathogen.

reassessment of water quality measures should be [Link]

performed in the municipality of Bacoor for the safety of dangerous-foodborne-pathogen/laboratory-typing-

consumers against waterborne diseases. methodsfor-diagnostic-of-salmonella-strains-the-

References old-organism-that-continued-chall

Al-Qadiri HM, Lu X, Al-Alami NI, Rasco BA (2010). Major Infectious Diseases (2014). US: CIA US.

Survival of Escherichia coli O157:H7 and [Link]

Campylobacter jejuniin Bottled Purified Drinking factbook/fields/[Link]

Water under Different Storage Conditions. J Food Momtaz H, Dehkordi FS, Rahimi E, Asgarifar A (2013).

Prot 10: 368. Detection of Escherichia coli, Salmonella species

BioMerieux® API Standard Procedure: API 20 E ID Kit and Vibrio cholera in Tap water and Bottled

Technical Manual. drinking water in Isfahan, Iran. BMC Public

Demirel R, Yimaz Sariozlu N, Kivanc M (2011). Health 13: 556.

Microbiological investigation of bottled mineral Mission (2012). Cavite: Dasma Water; Retrieved from:

and drinking water sold in Eskisehir (Turkey) [Link]

markets 1(2): 153-160. Pandey SR, Jegatheesan V, Baskaran K, Shu L (2012).

DES (2010). Causes of Positive Bacteria Results in Fouling in reverse osmosis (RO) membrane in

Water Samples. water recovery from secondary effluent: a review.

[Link]
 Francisco et al., 2015

Rev Environ Sci Biotechnol 11:125-145. Water Sanitation (2004). Water for Health-Taking

Philippines, Department of Health, Philippine Health Charge. Geneva: World Health Organization;

Statistics (2009). Leading causes of morbidity; (cited on 2014 February 5).

[cited on 2014 March 19]. [Link]

[Link] [Link]

Philippines, Department of Health, Philippine Health

Statistics (2009). Infant mortality ten (10) leading

causes; [cited on 2014 March 19].

[Link]

Philippines, Department of Health, Philippine Health

Statistics (2009). Leading causes of child mortality

causes; [cited on 2014 March 19].

[Link]

[Link]

Philippine National Standards for Drinking Water

(2007). Philippines: Department of Health [cited

2015 Jan].

[Link]

National-Standards-for-Drinking-Water-

2007#scribd

Profile of Maynilad (2009). Philippines: Maynilad Water

Services Incorporation; [cited on 2014 February

5]. [Link]

Sarwar G, Khan J, Iqbal R, Afridi AK, Khan A, Sarwar

R (2011). Bacteriological Analysis of Drinking

Water from Urban and Peri-urban Areas of

Peshawar. JPMI. 18(1).

Sources of water (2012). Cavite: Dasma Water;

[Link]

[Link]

Tortora GJ, Funke BR, Case CL (2013). Microbiology:

An Introduction. Singapore: Pearson Education

South Asia Pvt. Ltd. 10th Ed.

[Link]