Bacteriological analysis of piped water and purified drinking water in Dasmariñas city and Bacoor city (Philippines)

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1 INT J CURR SCI 2015, 17: E RESEARCH ARTICLE ISSN 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@dlsud.edu.ph 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: 14 th April 2015; Revised: 28 th May; Accepted: 14 th June; IJCS New Liberty Group 2015 Introduction Water is one of the natural resources needed by humans for their survival. It is used for preparation and consumption of food, washing and bathing. Aside from these, it may also serve as a mode of transmission for microbes like Escherichia coli, which produce toxins that cause gastrointestinal disturbances and Salmonella Typhi, which causes typhoid fever (Tortora et al., 2013). If the water used for human consumption does not undergo a well-processed treatment system, this can give a higher risk of causing diseases to consumers. Thus, infants and children who have low immune system will get higher chance of suffering from diseases such as diarrhea, cholera, and gastroenteritis (WHO, 2004). In the Philippines, water is treated in treatment plants and distributed by water concessionaires. The water source of Bacoor City, supplied by Maynilad uses Putatan Treatment Plant to treat raw water from Laguna Lake. It has fourteen units of microfiltration assemblies and six reverse osmosis assemblies (Maynilad, 2009). On the other hand, the water source of Dasmariñas City supplied by Dasmariñas Water District (DWD) treats its water by chlorination after filtration (DWD). The application of chlorine is used to disinfect water supply from possible contamination. This is to ensure that water being supplied to consumers is safe, clean and

2 potable. However, water could still be a source of infection after it has undergone several purification processes (Demirel et al., 2011). In 2009, there were 685 mortalities recorded of intestinal infectious disease in Region IV-A, which makes intestinal infectious disease as one of the leading causes of death in the region having a 4% increase compared to its previous year (DOH, 2009). Aside from diarrhea, studies showed that the ingestion of contaminated water with coliforms of Escherichia coli, Salmonella sp, Vibrio cholerae and other microorganisms can be the source of serious complications like enteritis, death, economic loss and also malaria, cholera, dysentery and hepatitis A (WHO, 2004). Acute watery diarrhea (AWD), Typhoid and Paratyphoid Fever are some of the leading causes of morbidity in the Philippines. There were 434,445 morbidities recorded in 2008 and 322,799 morbidities recorded in 2009 for AWD. This showed a decrease of 25% percent in total number of morbidities of AWD from 2008 to There were 11,119 morbidities recorded in 2008 and 6,279 morbidities recorded in 2009 per 100,000 populations. A decrease of 44% in total number of morbidity per 100,000 populations was from 2008 to 2009 (DOH, 2009). The infant mortality of diarrhea and gastritis of presumed infectious origin per 1000 live births in the Philippines showed a three percent increase from 2008 to There were 937 mortalities recorded in 2008 and 971 mortalities recorded in 2009 (DOH, 2009). The intestinal infectious disease is one of the leading causes of death in Region IV-A. There were 656 mortalities recorded in 2008 and were 685 mortalities recorded in This showed a four (4) percent increase of the intestinal infectious disease in Region IV-A from 2008 to 2009 (DOH, 2009). The child mortality of diarrheas and gastritis of presumed infectious origin per 100,000 populations in the Philippines from 2008 to 2009 showed a thirteen percent increase for ages 1 to 4, a constant child mortalities for ages 5 to 9 while no mortalities were recorded for ages 10 to 14 (DOH, 2009). Waterborne diseases threaten the human health even after the water has undergone modern techniques like disinfection, sanitation and water purification (Momtaz et al., 2013). Assessing the microbiological quality of piped water and purified drinking water is needed in order to monitor the occurrence of Escherichia coli and Salmonella Typhi along with other pathogens that cause waterborne diseases. This is to measure the number of occurrences of diarrhea, dysentery, enteritis and death especially here in the Philippines wherein our water resources are easily exposed to sewage and pesticides due to our agricultural activities. This study assessed the water quality of purified drinking water and piped water in Dasmariñas City and Bacoor City. Materials and Methods This study used a descriptive research design in assessing the water quality of purified drinking water and the piped water in Dasmariñas and Bacoor City. Heterotrophic plate count was used in determining the heterotrophic bacteria present. The selective plating was done for preliminary assessment while the Analytical Profile Index (API) kit was used in confirmation of the identity of isolates. Forty-eight (48) samples were collected within the limits of Dasmariñas City and Bacoor City. Sample collection: In total, forty-eight (48) samples were collected within the two cities. The water samples

3 consisted of 12 samples of piped water and 12 samples of purified drinking water were obtained from Dasmariñas and Bacoor, cities of Cavite. The water samples were collected randomly in Dasmariñas City and Bacoor City. One hundred milliliters (100 ml) of piped water was collected and placed in a sterilized bottle. One hundred milliliters (100 ml) of purified drinking water was bought from the local stores located within the two cities. All water samples were transported to the laboratory after the collection and processed immediately. Heterotrophic plate count The bacteriological assessment of water quality used the heterotrophic plate count method. Briefly, 1 ml of the water sample was mixed with liquefied R2A medium. It was allowed to solidify for 5 mins. After 5 mins, the plates were incubated at 37 o C for hrs. After incubation, the counts were determined and the results were expressed as cfu/ml (colony-forming unit per milliliter) (Zeenat et al., 2009). Preliminary Identification of Escherichia coli and Salmonella Typhi In the preliminary identification of E. coli and S. Typhi, selective plating method was used. The colonies from the HPC positive were streaked in eosin-methylene blue agar for E. coli (EMBA) and Salmonella-Shigella Agar for S. Typhi (Imen et al., 2012). Confirmation of the identity Isolates were identified using API 20 E (BioMerieux ) identification kit. Colonies were obtained from purified cultures in eosin-methylene blue agar (EMBA) and Salmonella-Shigella Agar (SSA) and grown in separate tubes containing 10 ml Nutrient broth inoculated at 37 0 C for 24 hrs. Reactions were read according to the reading table and identification or were obtained by referring to the analytical profile index (API) (Imen et al., 2012). Statistical analysis Colonial characteristics were observed in R2A, SSA, and EMBA plates. Such characteristics include size, form, margin, surface elevation, consistency and pigmentation. Analytical Profile Index (API) 20E ID kit was used to identify E. coli and S. Typhi, which uses 20 miniaturized biochemical tests. Color changes in every test were recorded. Chi square test was used to determine the difference between piped water and purified drinking water in relation to HPC. It was also used to determine the difference between source water of Dasmariñas and Bacoor City, Cavite. All statistical analysis was conducted using STATA 9.0 for windows (STATA Corp., College Station, Texas USA). Significance was defined as a probability of less than 0.5 (p< 0.05). Results and Discussion The assessment of bacteriological quality of drinking water for fecal contaminants is very important due to increasing diarrheal and typhoidal diseases in the Philippines (CIA US 2014). In Philippines, 322,799 cases were recorded for acute watery diarrhea in 2009 and still one of the leading causes of morbidity (DOH, 2009). This study was conducted to assess the microbiological quality of piped- and purified drinking water in Cavite province, Philippines. Table 1 shows the heterotrophic plate count of 24 piped water samples and 24 purified drinking water samples collected from Dasmariñas and Bacoor City. In Dasmariñas City, 33.3% (4/12) were observed in purified drinking water, while no sample rendered positive in piped water. In Bacoor City, both of the type of water samples showed positive

4 results. In purified drinking water, 33.3% (4/12) were observed, while in piped water, 58.33% (7/12) were positive to HPC. There are a higher number of heterotrophic plate counts for both piped water and purified drinking water in Bacoor City than in Dasmariñas City. The heterotrophic plate count indicates the contact of water with sewage or inadequate treatment or post treatment contamination (Sarwar et al., 2011). The purified drinking water collected randomly from Dasmariñas and Bacoor City undergoes Piped water Table 1. Heterotrophic plate count (HPC) of the water samples Samples Number samples Number w/ HPC positive (>500 cfu/ml) Dasmariñas 12 0 Bacoor 12 7 Purified drinking water Dasmariñas 12 4 Bacoor 12 4 Table 2. The presence of S. Typhi and E.coli 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 City, out of 12 purified drinking water samples brought from local water drinking stations, 4 were HPC positive and in Bacoor City, out of 12 purified drinking water samples brought from the local water drinking stations, 4 were HPC positive. The bacterial growth is higher in bottled water because plastic is more permeable to oxygen and extraneous vapors and because it also releases nutrients that increase bacterial growth. Bacterial adherence to bottle surfaces aid in its survival process (Al-Qadiri et al., 2010). Purified drinking water samples contain more coliforms than that of the piped water samples from both Bacoor and Dasmariñas Cites. This is due to the absence of disinfection treatment after bottling where an increase in bacterial content had sourced due to the revivable colony count (Demirel et al. 2011). The presence of microorganism after the water treatment is due to biological activity occurring in water treatment equipments wherein the filter media held together the bacteria and provides adequate food supply for the bacteria (DES 2010). In Dasmariñas City, the piped water undergoes filtration and chlorination (DWD). Our results showed the effectiveness of chlorination in water treatment process since there is no

5 HPC positive in the 12 piped water samples collected randomly in Dasmariñas City. Chlorination is used by water processing plants in water treatment. It shows to be effective for killing bacteria and viruses but not for some protozoan cysts (SDWF). While in Bacoor City, the piped water undergoes large-scale microfiltration and reverse osmosis (Maynilad). Our results showed that out of 12 water samples collected randomly in Bacoor City, piped water and purified drinking water in relation to heterotrophic plate count (HPC). This means that the water type does not affect the number of HPC. However, there was a significant difference (p=0.035) between the water samples from Bacoor and Dasmariñas City in relation to HPC. This means that the collection site determines the number of HPC which varies depending 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 of microbial bio-films. This is the biggest problem in membrane filtration because even with highly effective pre-treatment processes that can remove 99.9% of all microorganisms, there are still enough cells left which can colonize and form a bio-film causing flux decline and irreversible fouling. This is a result of formation of bio-films on membrane surfaces. Once bacteria attach to the membrane surfaces, they start to multiply and produce extracellular polymeric substances (EPS) to form a viscous, slimy, hydrated gel (Pandey et al., 2012). Thus, water quality in Bacoor City is polluted by fecal contaminants, leaking sewage lines, human and animal excreta flowing into open drain system, which is the common potential source of contamination in defective drinking water distribution system (Din et al., 2014). No significant difference (p>0.05) was observed between Table 2 shows the presence of S. Typhi and E. coli of 24 piped water samples and 24 purified drinking water samples collected from Dasmariñas and Bacoor City. The colonies from heterotrophic plate count (HPC) subjected to Salmonella-Shigella Agar and eosinmethylene blue agar were identified and observed. In Salmonella-Shigella agar, presence of black colonies (S. Typhi) was only observed in one sample from a purified drinking water collected at Dasmariñas City. Wherein eosin methylene blue agar, presence of metallic green colonies (E. coli) were found in both types of water samples (one in piped water and two from purified drinking water) collected from Bacoor City. No metallic green colonies (E. coli) found from any type of water samples collected from Dasmariñas City. Figures below show the S. Typhi and E. coli isolates. Colonies resembling S. Typhi and E. coli were confirmed

6 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 conform to S. Typhi and E. coli. For confirmation of the positive results from eosin methylene blue agar and Salmonella-Shigella agar, the isolates were subjected to BioMerieux API 20 E ID KIT. Table 3 shows the results of 21 tests for each isolate. At each test, the positive and negative colors are noted for assessing the identification of each isolates. The results are tabulated for their identification number. The identification number of 1aS1 is which is classified as S. Typhi. The 2cE2, 4cE1 and 2dE1 share the same identification number, which is classified as E. coli. The presence of fecal indicator bacteria, including coliforms, S. Typhi and E. coli, indicates the potential presence of enteric pathogens and related human health risk, as these microorganisms are commonly found in

7 mammalian intestines (Smith et al., 2014). Although most of these organisms do not cause illness to human, they can cause nuisance through generation of taste and odor or discoloration of drinking-water supplies (PNSDW 2007). Conclusion Assessing the water used for consumption is very important because this water source would determine the health condition of the consumers. The water quality according to the Philippine National Standards for Drinking Water (PNSDW) states that the water sample should be free from any pathogenic bacteria. Hence the samples, positive to HPC and S.Typhi and E.coli, were recognized as non-potable. Bacoor City has higher bacterial load for both piped water and purified drinking water than that of Dasmariñas s. This could mean that a reassessment of water quality measures should be performed in the municipality of Bacoor for the safety of consumers against waterborne diseases. References Al-Qadiri HM, Lu X, Al-Alami NI, Rasco BA (2010). Survival of Escherichia coli O157:H7 and Campylobacter jejuniin Bottled Purified Drinking Water under Different Storage Conditions. J Food Prot 10: 368. BioMerieux API Standard Procedure: API 20 E ID Kit Technical Manual. Demirel R, Yimaz Sariozlu N, Kivanc M (2011). Microbiological investigation of bottled mineral and drinking water sold in Eskisehir (Turkey) markets 1(2): DES (2010). Causes of Positive Bacteria Results in Water Samples. ctsheets/dwgb/documents/dwgb-4-2.pdf Din M, Ahmad Z, Aleem A, Pirkani GS, Mohammad A, Ahmad N (2014). Pathogens From Drinking Water; Isolation and antibiogram of pathogenic organisms from drinking water in Quetta city. Professional Med J 21(4): EPA (2005). Water Health Series: Bottled Water Basics. 816-K s_bottledwater.pdf Imen BS, Ridha M, Mahjoub A (2012). Laboratory Typing Methods for Diagnostic of Salmonella strains, the Old Organism That Continued Challenges. Salmonella-A Dangerous Food borne Pathogen. Major Infectious Diseases (2014). US: CIA US. factbook/fields/2193.html Momtaz H, Dehkordi FS, Rahimi E, Asgarifar A (2013). Detection of Escherichia coli, Salmonella species and Vibrio cholera in Tap water and Bottled drinking water in Isfahan, Iran. BMC Public Health 13: 556. Mission (2012). Cavite: Dasma Water; Retrieved from: Pandey SR, Jegatheesan V, Baskaran K, Shu L (2012). Fouling in reverse osmosis (RO) membrane in water recovery from secondary effluent: a review.

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