|Year : 2020 | Volume
| Issue : 1 | Page : 17-22
Bacteriological quality of water samples from Kochi, Southwest Coastal India and its implications
Sushma Krishna1, Sadia Khan1, Kavitha Dinesh1, S Aswathy2, Vinitha Viswanath1, Shamsul Karim1
1 Department of Microbiology, Amrita Institute of Medical Sciences, Cochin, Kerala, India
2 Department of Community Medicine, Amrita Institute of Medical Sciences, Cochin, Kerala, India
|Date of Submission||15-Apr-2020|
|Date of Decision||21-May-2020|
|Date of Acceptance||01-Jun-2020|
|Date of Web Publication||13-Aug-2020|
Dr. Sushma Krishna
St. Martha's Hospital, Nrupathunga Road, Bengaluru, Karnataka
Source of Support: None, Conflict of Interest: None
BACKGROUND: Coliforms and other bacterial indicators are known to be present in Community water samples causing faecal pollution of water. There is also a growing body of evidence demonstrating that the aquatic environmental microbes are drug resistant.
OBJECTIVE: The objective of this study was to evaluate the microbial quality of the water from tap, well and pond in urban households of Kochi, Kerala state.
MATERIALS AND METHODS: A total of 100 water samples (46 from well water, 45 from tap water and 9 from lakes and ponds) were analysed by the multiple fermentation tube method to determine the presumptive coliform count or the most probable number (MPN) of coliforms, and the isolates were identified using standard procedures, followed by susceptibility testing.
RESULTS: Eighty samples were positive for growth (76 grew coliforms, four samples grew non-pathogens). A total of 105 coliform isolates were grown in culture. The MPN numbers were noted to be >100 for over 50% of all the samples (n = 56), thus making the water unsatisfactory for drinking purposes. The highest number of organism isolated was Klebsiella pneumonia (n = 55), followed by Enterobacter spp (n = 34) and Escherichia coli (n = 16). Well water had the highest proportion of all three of these organisms. Up to 26%–40% resistance was seen to amoxicillin–clavulanic acid and 5.4%–5.8% of resistance was noted to third-generation cephalosporins, while two isolates were extended-spectrum beta-lactamase-producing Enterobacteriaceae (CTX-M and TEM types).
CONCLUSION: The city municipal water authorities need to adopt more aggressive treatment/disinfection practices to combat high coliform contamination. The bacteriological quality of well water at the source needs to be monitored. Meanwhile, continued adequate home purification is suggested for drinking water. The city water does not pose a threat of antibiotic resistance for now. Environmental sampling should be given equal priority as clinical sampling in the coming days.
Keywords: Antibiotic resistance, coliforms, Kochi, water
|How to cite this article:|
Krishna S, Khan S, Dinesh K, Aswathy S, Viswanath V, Karim S. Bacteriological quality of water samples from Kochi, Southwest Coastal India and its implications. J Acad Clin Microbiol 2020;22:17-22
|How to cite this URL:|
Krishna S, Khan S, Dinesh K, Aswathy S, Viswanath V, Karim S. Bacteriological quality of water samples from Kochi, Southwest Coastal India and its implications. J Acad Clin Microbiol [serial online] 2020 [cited 2022 Jan 27];22:17-22. Available from: https://www.jacmjournal.org/text.asp?2020/22/1/17/291896
| Introduction|| |
India faces the challenge of waterborne infectious diseases as a major public health problem. Enteric pathogens such as Diarrhoegenic Escherichia coli, Salmonella Spp, Shigella Spp, Vibrio cholerae and Entamoeba histolytica contribute to a major burden and continue to affect the biological quality of water. Improper sanitation, unhygienic management of water supply, lack of awareness among users, industrial and domestic sewage runoffs affect the quality of water. Diarrhoea is the third-most common cause of death in children under the age of five, responsible for 13% deaths or about 300,000 children in this age-group in India each year. Most enteric pathogens can be cultured in the laboratory from stool samples of symptomatic patients. The indicator organisms or the bacterial indicators causing faecal pollution of water, such as coliforms, fecal Streptococci and Clostridium perfringens, can also be detected in the laboratory from environmental water samples by standard laboratory techniques. There is also a growing body of evidence demonstrating that the aquatic environmental microbes are drug resistant. The causes have been identified as the hospital, municipal and aquaculture effluents, which are discharged into the water bodies. Humans, domestic livestock, pets, wild animals and birds are all intestinal reservoirs of coliforms, which are spread by the faecal-oral route. Transmission to humans from these reservoirs may occur in a variety of ways, including contact with other humans or animals, manure or sewage, or contaminated meat/poultry products, vegetables, raw milk or untreated water.,,, A significantly high percentage of extended-spectrum beta-lactamases (ESBLs) and the infamous (New Delhi Metallo-beta-lactamases, hydrolyzing carbapenems such as imipenem, meropenem) producing strains have been known to be circulating in the Indian sub-continental community in drinking water and sewage samples, and have also been claimed to have disseminated globally, impacting international travel.,, The objective of this study was to evaluate the microbial quality of the water from the tap, well and pond in urban households of Kochi, the Western part of Ernakulum, a cosmopolitan city and the business hub of the Southern Indian state of Kerala.
| Materials and Methods|| |
A total of 100 water samples covering an area of over 1500 sq km were randomly collected from various locations of the Cochin district over one year period (November 2011–November 2012) and were processed at the National Board Accredited Microbiology Laboratory (NABL) of a 1200 bedded University Teaching Hospital at Cochin. The district is an urbanised seaport and stretches for around 3068 sq km with a population of 601,574 (2011 National census data). The state mostly houses urban and semi-urban population, and it is believed that the city water supply to the households is of good quality due to the regular disinfection, which is taken up by the State Water Supply Authority Board regularly. The house inmates were informed and consented for water collection. Data were collected about the location from which the sample was randomly collected, its source, the use of the water as for domestic or non-domestic purposes, the total number of people who consumed it, and the methods of purification used in drinking water samples (if any) was recorded. Information on the socioeconomic status of the households was, however, not gathered.
Sample collection, handling and transport
Glass bottles with fitting screw caps of the capacity of 200 ml were used for water sampling. Covered with aluminium foil, the bottles were sterilised in a hot air oven at 160°C for 1 h. Care was taken to prevent accidental contamination of the water during its collection. Bottles were transported to the processing within 4–6 h in a tightly-closed cooling carry-box with ice packs at 4°C.
Samples from house taps
Any external fittings were removed before collecting water, and the outside nozzle of the tap was cleaned. Some amount of water was allowed to run for a minute after turning on the tap to flush any stagnant water in the service pipe. Then the tap was sterilised using the spirit lamp or by igniting a piece of cotton-wool soaked in methylated spirit with a pair of tongs close to the nozzle until the whole tap was unbearably hot to touch. Again some amount of water was left to run for a few seconds so that the tap becomes cool and a gentle flow of water was filled and capped. Using a water-proof marker, the labelling of the bottle was done. Forty-six samples were collected thus. Tap water from stored containers by the households was not regarded as a sample for the study.
Samples from wells
For an open well, a weighted bottle was used to collect the sample by tying the sterile sample bottle onto a weighted length of rope. The cap of the bottle was then removed aseptically, and the bottle lowered into the well to a depth of about 1 meter. When no more air bubbles were seen on the surface, the bottle was raised out of the well and its cap was replaced carefully and the bottle was labelled. Forty-five samples were collected thus (including four from bore-well).
Samples from pond, lakes, field streams
After aseptically removing the cover and cap of the sterile sample bottle, the mouth of the bottle was placed upstream (toward the flow of water). Its neck was plunged downwards about 30 cm below the water surface and then, the neck of the bottle was tilted slightly upwards to let it fill completely before carefully replacing the cap and cover and then, the bottle was labelled with the number. The 9 water bodies sampled included-Periyar river (n = 1), Amrita Lake (n = 1), Kumbalangi river (n = 1), Varapuzha river (n = 1), Aluva canal water (n = 1), Piravum pond (n = 1), Karukutty pond (n = 1), Ponekkara temple pond (n = 1) and Chellanam river (n = 1). There are no major sewage plants with affluent discharge into or in the vicinity of these water bodies, but animal grazing seen. This water was not used for drinking purposes.
Presumptive coliform count (multiple tube fermentation technique)
An estimation of the number of coliform bacilli in a water supply was made by adding varying quantities of the water (from 0.1 [1 / 10th of dilution of sample, respectively], 1, 10 ml and 50 ml) to Mac Conkey broth (with an indicator of acidity). Using sterile graduated pipettes, the following quantity were mixed: For 1 bottle of 50 ml water: 50 ml double strength medium, for 5 bottles of 10 ml of water: 10 ml double strength medium, for 5 bottles of 1 ml of water: 5 ml of single strength medium and for 5 bottles of 0.1 ml of water: 5 ml single strength medium each. Tubes contained Durham tubes to show the production of gas. Acid and gas formation (a 'positive' result) indicates the growth of coliform bacilli. The bottles were incubated at 37°C and examined after 18–24 h. Any remaining negative bottles were re-incubated for another 24 h and if acid and gas developed, they too were regarded as positive. In reporting the results of the presumptive test, reference was made to Mc Crady's probability tables. More than 10 coliform per 100 ml in un-piped rural supplies and >3 per 100 ml in non-chlorinated pipe supplies were considered unsatisfactory for drinking as per International WHO standards. Positive samples were sub-cultured on Mac Conkey agar plates and kept for overnight incubation for about 18–24 h (the culture media underwent strict sterility evaluations before inoculations). Identification of Enterobacteriaceae was made based on standard biochemical identification tests recommended. Antibiotic susceptibility testing for antibiotics was done by Kirby-Bauer's disc diffusion and ESBL detection by double disk diffusion test based on the Clinical Laboratory Standards Institute guidelines. The Minimum Inhibitory Concentration to various antibiotics in isolates with ESBL activity (if any) were further determined by Vitek-2 compact (Biomerieux clinical diagnostics, France, headquarters: New Delhi, India)., The ESBL-positive isolates were subjected to multiplex polymerase chain reaction (PCR) for gene detection.
| Results|| |
A total of 100 water culture samples were analysed during the study period. Forty-six samples were from well water, 45 were from tap water and 9 were from lakes and ponds. Of the hundred samples, 80 samples were positive for growth (76 grew coliforms, 4 samples grew non-pathogens). The prevalence rate was thus 76% for coliforms [Table 1]. A total of 105 coliform isolates were grown in culture. The MPN numbers were noted to be >100 for over 50% of all the samples (n = 56), thus making the water unsatisfactory for drinking purposes. Out of 36 water samples exclusively used for drinking, 40 coliforms were grown. Purified drinking water also grew 7 coliforms out of 29 samples [Table 2]. Among the localities in Kochi, Edappally area had the maximum coliform contamination (36 samples out of 52). The highest number of organism isolated was Klebsiella pneumonia (n = 55) followed by Enterobacter spp (n = 34) and E. coli (n = 16). Well water had the highest proportion of all three of these organisms. All the results are tabulated below. Two (1.9%) out of 105 isolates were found to be ESBL producing Enterobacter Spp. Most of the isolates remained susceptible to the majority of commonly tested antibacterial agents [Table 3]. Up to 26%–40% resistance was seen to Amoxicillin-clavulanic acid and 5.4%–5.8% of resistance was noted to the 3rd generation cephalosporins while two isolates were ESBL-producing Enterobacteriaceae. The ESBLs producers were from well water (n = 1) and pond water (n = 1). Among the localities in Kochi, Edappally and Varapuzha (local area map attached) area had one ESBL producing organism each. The ESBL genes were detected by PCR according to which, the Enterobacter spp isolated from the pond water in Varapuzha contained CTX-M type and the Enterobacter spp isolated from well water in Edappally contained TEM type. No illness was reported from people all over whoever had consumed the water. Water treatment details could not be elicited.
|Table 2: Coliforms isolation in the purified and unpurified drinking water|
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|Table 3: Antimicrobial susceptibility profile of Gram-negative bacteria isolated from water samples (number sensitive and percentage resistant)|
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| Discussion|| |
Coliform contamination rates in drinking water are known to be high in developing countries where environmental sanitation and hygiene are still inadequate and the same has been previously noted in the district. The present study shows 58% of drinking water to be contaminated, making it unsatisfactory for drinking purposes. Over 60% of the well water samples had coliform contamination, mostly attributed to the watershed erosion due to flooding and seepage of sewage and swamps. Traditional houses with wells in the backyard, in close proximity to underground septic pits, could possibly have been contaminated as described in Mukhopadhyay et al. However, the further pathogenic potential of these isolates has to be evaluated before declaring them as major threats. Satisfactory community awareness with high literacy in the state (99%) coupled with health indicators comparable to the West, is reflected in the fact that over 83% of the study population who had coliform positive water employed additional purification methods before drinking. The urban/semi-urban population seldom uses open river water for drinking purposes. The danger of waterborne diseases in the community as a result of drinking water drawn from non-potable sources like streams may be significant in other parts of the country.
The organisms isolated in the study did not exhibit significant resistance to third-generation cephalosporins and other higher antibiotics. Like most of the tertiary care hospitals in the country, our hospital's ESBL Enterobacteriaceae rates are in the range of 62%–100%, termed as epidemic proportions (SARI 2006, member hospital, ICMR-ESBL study surveillance). The ESBL community prevalence rate varies with geographic locations, different practices followed in developing and developed countries and the type of water sampled. In comparison with the ESBL prevalence rates of 2.5%–47.3% in water samples from across the world,,,,, our rates are lower, possibly due to the presence of bacteriophages in the tested water, needing further confirmation. If the study results with disproportionately lower ESBL community rates are regarded as a positive finding despite a small sample size, it likely points out that the source of transmission may not be water. Data regarding ESBL carriage in isolates from the community is still emerging in the country, and the results of this study can be used as a reference for further studies in this geography. Low resistance is not equivalent to no resistance, and the low ESBL rates may also be attributed to the low intestinal carriage of humans and animals in this place, although grazing animals such as cattle, local birds and other fauna had access to water bodies. Intestinal carriage of ESBL E. coli in humans and animals is one of the transmission paths by which antimicrobial resistance spreads in the community where faecal matter mixes with river micro-biota. Although no effluents and other wastewater joined the rivers sampled, the water from the ponds, stream and the like may have been used for farming and gardening purposes (one of the water streams in our study was used for growing paddy), which again forms a sub-link in human transmission cycle.,
Since one of the ESBL isolates was from the river water, the importance of river water as a reservoir of antimicrobial resistant genes and resistant bacteria should not be underestimated. However, effluent discharge of hospitals with high 3rd generation cephalosporin usage was clearly non-contributory in this study. In this context, screening for ESBL intestinal carriage in patients at admission to multi-speciality hospitals may hence be considered to determine the actual carriage rates of community. Preliminary on-going molecular studies in our hospital reveal a staggering number of 45%–50% intestinal carriage rates (unpublished data).
Armstrong et al. 1981 had found that drug-resistant organisms were more common in drinking water than in untreated water sources. They found that the treatment of raw water and its subsequent distribution select for bacteria exhibiting the drug-resistant phenotype and is attributed to shedding and living in pipelines. Bio-film formation in pipelines, long storage of standing supplies and leaking pipes may also contribute to the higher rates. No ESBL producers were, however, found in the chlorinated tap water supplies. Links of transmission such as through sea-food (staple diet of coastal Kerala) where intensive use of antimicrobial agents in aquaculture creates reservoirs of drug-resistant bacteria and transferable resistance genes in fish pathogens with subsequent dissemination by horizontal gene transfer and reach human pathogens remains unexplored.
| Conclusion|| |
Although city water is heavily contaminated with coliforms, it does not seem to contribute to or worsen the antibiotic resistance scenario of the environment. The results come as a relief but should be viewed as an early indicator of the impending problem of resistance (ESBL hospital-community transmission of infections) in the local community water samples. Bacteria carrying resistance genes for many antibiotics are moving beyond the clinic into the community, infecting otherwise healthy people with untreatable and frequently fatal infections. At the same time, community ESBL transmission may have other unknown reservoirs other than drinking water. For now, continued adequate home purification is suggested. The municipal authorities need to adopt more aggressive treatment/disinfection practices to combat high coliform contamination. Environmental sampling should be given equal priority as clinical sampling in the coming days.
We are grateful to Dr. Anand Manoharan and Ms. Geethika Barla, Research wing, IDTRC, CMC, Vellore, for the molecular help. We also thank the families from Cochin who contributed the water samples for the study.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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