My Scientific Blog - Research and Articles: April 2011

Hishila Sujakhu¹and Upendra Thapa Shrestha^{1, 2}

Khwopa College, Tribhuvan University, Dekocha, Bhaktapur.

Kantipur College of Medical Sciences, Tribhuvan University, Sitapaila, Ktm.

Corresponding author: Upendra Thapa Shrestha, Faculty, Khwopa College, email: upendrats@gmail.com

ABSTRACT

Forty water samples were collected from Bhaktapur Municipality ward no. 10, one of the most water borne diseases suffering regions, following standard methods described by APHA. All samples were transferred to Microbiology laboratory of Khwopa College, Dekocha, Bhaktapur and preceded immediately for Microbial analysis. The coliform density in the water samples were determined by Most Probable Number (MPN) method followed by microscopy, colonial morphology and biochemical characterization. Subsequently, the presence of Vibrio cholerae, a causative agent of Cholera was analyzed in the same samples by using Thiosulphate citrate bile-salt sucrose (TCBS) agar, a selective media for Vibrio sps. followed by oxidase test. Among forty water samples, 87.5 percent water samples contained coliforms and half of which contained feacal coliforms, Escherichia coli and remaining 12.5 percent water samples contained no coliforms. Vibrio cholerae were also isolated from two water samples.

Key words: MPN, Coliforms, TCBS agar, Vibrio cholerae and Oxidase test.

INTRODUCTION

Water is essential for life: both in terms of quantity and quality. Every year, approximately 3.4 million people die due to water-related diseases with the majority being young children under the age of five. Diarrheal diseases alone account for 2.1 million deaths per year. Other types of diseases associated with poor water quality include cholera, typhoid, arsenic poisoning, schistosomiasis or “snail fever”, and trachoma, which cause blindness (Dies et. al, 2003). Lack of hygiene knowledge, unsafe drinking water and sanitation are major causes of Diarrhoea with highest number of morbidity and mortality in the developing countries including Nepal (WHO, 1993).

Water borne diseases outbreaks (WBDO’s) occur when drinking water becomes contaminated by microbial pathogens or chemicals. Typically, WBDO’s are caused by microbial pathogens such as bacteria, viruses or protozoan. These pathogenic organisms are transmitted via the fecal oral route. This means that the drinking water supply has somehow been contaminated by fecal materials from humans or other warm blooded mammals (Gleick, 2002). Among bacterial outbreaks, cholera is perhaps, currently the most characterized of the major waterborne infections, but less of a threat compared to Enterobacteriaceae. The primary causative agent of cholera, Vibrio cholerae, is part of the normal bacterial flora of estuaries and most strains are not harmful to humans. Two strains are associated with establishment of diarrhoeal disease in humans, serogroups _O1 and _O139. These express the cholera enterotoxin (CT). Symptoms can vary from the most severe cholera gravis that is lethal, to inapparent infection (Woodall, 2010). Analysis of global trends in the incidence of cholera by five-year periods shows a steady increase since the beginning of the millennium. From 2004 to 2008, a cumulative total of 838,315 cases was notified to WHO, compared with 676,651 cases between 2000 and 2004, representing a 24% increase in the number of cases reported for this most recent five-year period as described in Cholera, 2010.

Many cities of Nepal including Bhaktapur municipality are continuously suffering a severe drinking water supply crisis, particularly in the dry seasons of every year. The drinking water supply in the city is intermittent. It has the poorest drinking water and sanitation coverage for its population among three districts in Kathmandu valley and a large percentage of its drinking water contains faecal coliforms (Diwakar et. al, 2008). Several bacterial, protozoal and viral water borne diseases have posed serious public health problem here. Diarrhoeal disease due to Enterobacteriaceae and Cholera are the two most prevalent water borne infections in the city. The explosive outbreaks of these diseases are the prime cause of death of thousands every year.

Therefore, in this research, our prime focus is to study about the occurrence of coliforms (especially faecal coliforms) and V. cholerae in drinking waters from outbreak regions of the city so that we can report and recommend those people who are continuously drinking the contaminated water.

METHODOLOGY

Sampling method and processing: The study was carried out in Bhaktapur municipality ward no. 10, one of the most outbreak regions for water borne diseases. Water samples for bacteriological testing were collected from main supply, household storage, stone tap and outbreak reported houses in sterile bottles by standard methods as mentioned in APHA and transported immediately for study. All the samples collected from those sources were used by people for direct drinking without prior treatment. Small aliquots of sample were enriched in alkaline peptone water for isolation of Vibrio spp. (Monica, 1998 and Chakraborty, 2001). Microbiological analysis of water samples was conducted in Microbiological Laboratory of Khwopa College, Dekocha, Bhaktapur. To minimize the error in results, samples were collected two times from same sites in the random period of intervals. So, total samples were two times the total number (2x40) =80. This was done to prevent the accidental results that might arise by any cause.

Microbial examination of water sample: Microbial examination of water samples were done by Most Probable Number (MPN) method as described by APHA for estimation of coliform and faecal coliform density, Escherichia coli. Detection of V. cholerae was done by the enrichment of water samples on alkaline peptone water, followed by isolation of the typical organism on selective medium, Thiosulphate Citrate Bile-salt Sucrose (TCBS) agar. Suspected Vibrio colonies were picked and subjected to biochemical tests including oxidase test to identify V. cholerae (Monica, 1998).

RESULTS

Among forty water samples, 37.5 % contained less than 30 coliforms per 100 ml, most of which contained 23 coliforms. 12.5 % water samples were found to be excellent containing no coliforms. While 17.5 % water samples contained less than 10 coliforms most of which contain less than 5 and only two samples were populated with 9 coliforms. 10% water samples contained 43 coliforms and similar percent of water samples were highly saturated with maximum number of coliforms equivalent to 1100. In remaining water sample, coliforms density of 93, 120, 150, 210, and 460 were found to be distributed equally which was equivalent to 2.5 % in each sample size. The results of presumptive test are given below in Table-1 and Figure-1.

Table-1: Results of Presumptive test.

Sample code	Total Coliform density= MPN/100ml	Faecal coliforms, E. coli
1. Block-tap water	28	+
2. Block-tap water	20	+
3. Block-tap water	150	+
4. Block-ground water	460	+
5. Block-tap water	1100	+
6. Block-tap water	210	+
7. Block-tap water	1100	+
8. Block-tap water	1100	+
9. Block-tap water	1100	+
10. Block-tap water	3	-
11. Block-tap water	0	-
12. Block-tap water	3	-
13. Block-tap water	43	+
14. Block-tap water	23	+
15. Block-tap water	9	+
16. Block-tap water	120	+
17. Block-ground water	9	+
18. Block-tap water	0	-
19. Block-tap water	0	-
20. Block-tap water	3	-
21. Block-ground water	3	+
22. Block-tap water	4	-
23. Block-ground water	43	+
24. Block-tap water	0	-
25. Block-tap water	23	-
26. Block-tap water	23
27. Block-tap water	23	+
28. Block-tap water	0	-
29. Block-tap water	23	-
30. Block-tap water	23	-
31. Stone tap water	93	-
32. Block-tap water	43	-
33. Block-tap water	23	-
34. Block-tap water	23	-
35. Block-tap water	23	-
36. Block-tap water	23	-
37. Block-tap water	23	-
38. Block-tap water	43	-
39. Tank water	23	-
40. Block-tap water	23	-

Note: Figures are given below.

Half or 50 % water samples processed in this experiment showed typical colonies with green metallic sheen morphology on EMB agar indicating the presence of E. coli. Further. Eighteen samples (45 %) containing E. coli were finally confirmed by positive indole and methyl red tests. However remaining 55 % water samples were free of faecal contamination i.e absence of E. coli (Figure-2).

Vibrio cholerae were isolated from two water samples (sample codes 16 and 27) which were collected from outbreak reported houses (Figure-2).

DISCUSSION

As samples were collected two times from same sites in the random period of intervals, the total samples were two times the total number (2x40) =80. This was done to prevent the accidental results that might arise by any cause. The results obtained for every two samples from each site were tallied and found to be reproducible. Hence the results shown for forty water samples were the representative data of whole samples (eighty samples). Among the forty water samples, 87.5 % (35 water samples) contained coliforms and half of which contained feacal coliforms and remaining 12.5 % (5 water samples) contained no coliforms. As presumptive test is only a preliminary test which is used for enrichment of coliforms in lactose media, the presence of gas production doesn't confirm the presence of coliform. Besides coliforms, some Gram positive bacteria like Bacillus, Streptococcus, Clostridium and Lactobacillus also give false positive result (Goel, 1997). The positive gas production in presumptive test were further confirmed by inoculating the samples in 2 % Brillliant Green Lactose Bile Broth (BGLB) and on Eosin Methylene Blue (EMB) agar, both of which are selective for coliforms. The typical colonies on EMB media finally confirmed the presence of faecal coliforms. About 50 % of water samples collected was found to be contaminated with faecal coliforms. The positive indole and methyl red tests add more to identify E. coli. According to WHO Guidelines for drinking-water quality, the drinking water should not contain any E. coli (WHO, 2003 and WHO, 2008). Therefore those water samples are not portable without prior disinfection by any physical and chemical methods. As mentioned in Dewakar et. al, 2008 and Wagle, 2009, the drinking water quality of valley region is still very poor.

The yellow colonies from many water samples show the presence of Vibrio species as most of them give yellow color on TCBS media which is a selective for V. cholerae and other Vibrio spp. of faecal origin. Colonies suspicious for V. cholerae appeared on TCBS agar as yellow, shiny colonies, 2 to 4 mm in diameter. The yellow color is caused by the fermentation of sucrose in the medium. Sucrose-nonfermenting organisms, such as V. parahaemolyticus, produce green to blue-green colonies (Kay et. al, 1994). However all V. cholerae are not pathogenic. They are part of the normal bacterial flora of estuaries and most strains are not harmful to humans. Only two strains are associated with establishment of diarrhoeal disease in humans, serogroups _O1 and _O139. Therefore the yellowish colonies from TCBS media should be further analyzed by oxidase test in order to identify the virulent strains. The sucrose fermenting colonies were again carefully inoculated on alkaline peptone medium. The fresh growth of organism was used for oxidase test. Neither colonies from Nutrient agar nor that from TCBS was used. Due to lack of salts, nutrient agar does not allow optimal growth of V. cholerae. Likely TCBS agar may yield either false-negative or false-positive results (Kay et. al, 1994 and CDC, 1994). In a positive reaction, the bacterial growth becomes dark purple immediately. The positive oxidase strains are virulent ones. Therefore in this work the evidence of two oxidase positive isolates shows that the water samples contain pathogenic strains. But the contamination is not at source. The possible source may be due to seepage, and cross contamination into distribution pipe lines.

CONCLUSION

The drinking water quality at that region was poor and not portable. Similarly the detection of V. cholerae in two drinking water samples indicates that cholera outbreak may occur in that region at any time in future. So people should disinfect the water by using feasible physical and chemical methods before drinking.

ACKOWLEGEMENT

We express our sincere gratitude to Uttam for laboratory assistant and all people who help us providing information and water samples.

REFERENCES

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