Journal of Infection and Public Health (2012) 5, 257262

Microbial quality of well water from rural and

urban households in Karnataka, India: A
cross-sectional study
Chiranjay Mukhopadhyay , Shashidhar Vishwanath, Vandana K. Eshwara,
Shamanth A. Shankaranarayana, Afrin Sagir

Department of Microbiology, Kasturba Medical College, Manipal University, Manipal 576104, India

Received 14 May 2011 ; received in revised form 25 February 2012; accepted 8 March 2012

KEYWORDS Summary
Escherichia coli; Objective: The objective of this study was to evaluate the microbial quality of the
Fecal coliforms; well water used as a drinking source in urban and rural households.
Most probable number; Methods: A total of 80 household well water samples were analyzed by the multi-
Total coliforms; ple fermentation tube method to determine the presumptive coliform count/most
Water quality probable number of coliforms, and the isolates were identied using standard pro-
cedures, followed by susceptibility testing.
Results: Fecal indicator organisms, including Escherichia coli and Enterococcus spp.
were isolated from 22 (27.5%) samples, and the majority (92.5%) of the water sources
were contaminated with coliforms. A total of 170 bacterial isolates were obtained,
including coliforms (70%), Enterococcus spp. (1.8%) and saprophytes (28.2%). A sig-
nicant number of isolates were multi-drug resistant, which is a cause of concern. A
comparison of the microbial quality of the water between urban and rural households
revealed no signicant differences.
Conclusion: It might be prudent to monitor the bacteriological quality of well water
at the source in addition to resistance proles of the isolates.
2012 King Saud Bin Abdulaziz University for Health Sciences. Published by Elsevier
Ltd. All rights reserved.

Corresponding author. Tel.: +91 9845513057;
fax: +91 820 2571927.
E-mail addresses: chiranjay@yahoo.co.in
(C. Mukhopadhyay), drshashidharv@gmail.com
(S. Vishwanath), vandanake@yahoo.co.in (V.K. Eshwara),
shamanth.adekhandi@gmail.com (S.A. Shankaranarayana),
afreen.rini@gmail.com (A. Sagir).
Introduction
Infectious diseases caused by pathogenic bacte-
ria, viruses and parasites are the most common
and widespread health risk associated with drink-
ing water [1]. Nearly one-tenth of the global disease
burden could be prevented by improving the water

1876-0341/$ see front matter 2012 King Saud Bin Abdulaziz University for Health Sciences. Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.jiph.2012.03.004
258
supply, sanitation, hygiene and the management of
water resources [2].
Water quality is affected by fecal matter, domes-
tic and industrial sewage and agricultural and
pasture runoff, in addition to a lack of aware-
ness and education among the users [3]. The
detection of bacterial indicators in drinking water
suggests the presence of pathogenic organisms that
are sources of waterborne diseases [4]. Indica-
tor microorganisms survive better and longer than
pathogens, with uniform and stable properties, and
may be easily detected using standard laboratory
techniques [5]. These indicator organisms include
Escherichia coli, thermotolerant (fecal) coliforms,
total coliforms, fecal streptococci and Clostridium
perfringens [6]. The two methods commonly used
to detect coliforms in water include the multiple
fermentation tube technique and the membrane
lter technique [7].
In coastal Karnataka, well water is an impor-
tant source of drinking and household water in both
rural and urban areas. Studies [3,4,817] assessing
the microbiological quality of drinking water have
found varying rates of contamination (0100%) with
fecal coliforms and other heterotrophic bacteria.
We conducted a cross-sectional study to analyze
and compare the microbiological quality of well
water in rural and urban households.
Methods
This pilot study was conducted in Udupi taluk
(population0.41 million) in Southern Karnataka,
India, over a period of two months between July
and August of 2009. Forty households each from
rural and urban areas were selected through simple
random sampling. Rural area refers to a place tured on MacConkey agar and incubated at 37 C
with a population of less than 5000 people, a popu-
lation density of less than 400 people per sq. km
and more than 25% of the male working popula-
tion engaged in agricultural pursuits. It is widely
believed that urban well water sources are of
good quality due to the availability of disinfectants
and awareness of the need for disinfecting water
wells.
Informed consent was obtained from the head
of each household before the water sample was
collected. Well water sources (dug wells) used as
the main source of drinking and household water
were included in the study, and wells that were
not in use or wells that were declared unt for
use were excluded from the study. All of the wells
screened were used by single families. Municipal
water sources or water from stored containers was
not included in the analysis.
C. Mukhopadhyay et al.
Sample collection
Following World Health Organization (WHO) guide-
lines [18], clean, heat-sterilized bottles of 200 ml
capacity were used for the water collection. A stone
of a suitable size was attached to the sampling bot-
tle using a piece of string. The bottle was opened
and lowered into the well; the bottle was com-
pletely immersed in the water, without touching
the sides of the well and without hitting the bot-
tom or disturbing any sediment. The bottle was
lled and then removed by rewinding the string.
Approximately 2030 ml of water was discarded to
provide sufcient airspace to allow shaking before
the analysis to achieve a homogenous dispersion
of the bacteria. After collection, the bottles were
labeled with complete details, including the source
of the water, the sample site, the address, and the
date and time of collection, and delivered (within
2 h) to the laboratory in a light-proof insulated
box containing ice packs. Before sampling the well
water, 45 drops of aqueous sodium thiosulphate
solution (100 g/l) was added to the sampling bot-
tles to neutralize any residual chlorine. Because
a complete history of chlorination (quantity, time
since last chlorination) could not be elicited, all
the sources were neutralized with sodium thiosul-
phate soon after collection, regardless of the prior
chlorination status.
Method of analysis
The water samples were processed using the mul-
tiple fermentation tube method to determine the
presumptive coliform count/most probable num-
ber (MPN) of coliforms based on standard methods
[19]. Suspensions from positive tubes were subcul-
for 2448 h. The resulting colonies were identi-
ed following standard operating procedures [20].
The antimicrobial testing of the isolates against
commonly used antibiotics was performed using
Kirby-Bauers disc diffusion method and was inter-
preted according to Clinical Laboratory Standards
Institute (CLSI) guidelines [21]. The detection of
extended-spectrum beta-lactamase (ESBL) produc-
tion was performed with a phenotypic method
using a double disc synergy test [22]. The micro-
bial quality of the water samples was assessed
based on WHO guidelines [6]. The results of rural
and urban areas were statistically compared using
the Chi-square test of association. The testing
of the water samples was performed according
to standard operating procedures, which were
strictly followed in the pre-analytical, analyti-
cal and post-analytical phases. Analytical quality
Microbial quality of well water 259

Table 1 Isolation rates of indicator bacteria from water samples.

Bacteria Urban Rural Total no. of samples Percentage
Escherichia coli 12 07 19 23.75
Fecal streptococci 00 03 03 3.75
Total coliform bacteria 39 35 74 92.5

control measures, including duplicate sample test-
ing, were performed. The culture media were
subjected to sterility and performance evaluations
before the samples were inoculated.
Results
The present study investigated the quality of house-
hold water from 80 samples collected at the source
in rural and urban communities. The fecal contam-
ination rate, as indicated by the growth of E. coli
or Enterococcus spp., was 27.5% (22 samples: 12
urban and 10 rural). Total coliforms were found
in 74 (92.5%) household water sources (urban39,
97.5%; rural35, 87.5%) [Table 1].
A total of 44 (55%) well water sources showed
the absence of E. coli and Enterococcus spp. and
the presence of 10 coliforms per 100 ml. Nine
(11.3%) samples without E. coli or Enterococcus
spp. had lower coliform counts (<10 coliforms/ml).
The absence of E. coli, fecal streptococci and
total coliforms was found in only 5 (6.3%) sam-
ples [Table 2]. A total of 48 (21 urban and 27
rural) samples showed a high MPN of >180. However,
the statistical analysis (Chi-square test of associa-
tion) did not reveal any signicant difference in the
quality of the water samples between the rural and
urban households (P = 0.39).
A total of 170 bacterial isolates were obtained
and included coliforms (119, 70%), Enterococ-
cus spp. (3, 1.8%) and saprophytic bacteria (48,
28.2%). The coliforms isolated included Klebsiella
spp. (48, 28.2%), Enterobacter spp. (30, 17.6%),
Escherichia coli (19, 11.2%), Proteus spp. (14, 8.2%)
and Citrobacter spp. (8, 4.7%). The environmental
saprophytes Pseudomonas aeruginosa (36, 21.2%)
and Acinetobacter spp. (8, 4.7%) were also isolated
from a signicant number of samples [Table 3].
Although the majority of the 119 coliform strains
were susceptible to commonly used antibacterial
agents, a signicant resistance was observed among
the isolates to beta-lactam antibiotics. In total, 91
(76.5%) strains were resistant to ampicillin, and
26 (21.8%) and 15 (12.6%) strains were resistant
to second- and third-generation cephalosporins,
respectively. Three (2.5%) isolates were resistant
to gentamicin, netilmicin and uoroquinolones. All
of the coliform strains were susceptible to amikacin
[Table 4].
Multi-drug resistance was observed in a signif-
icant number of the isolates. Twenty-ve (21%)
coliform strains and 2 (66.7%) Enterococcus spp.
were resistant to more than two classes of

Table 2 Distribution of indicator bacteria and saprophytes in urban and rural water samples.
Isolates Urban Rural Total
E. coli 2 1 3
E. coli and one coliform 3 1 4
E. coli and two coliforms 2 2 4
E. coli and a saprophyte 1 0 1
E. coli, a coliform and a saprophyte 4 3 7
Fecal streptococci 0 1 1
Fecal streptococci and a coliform 0 1 1
Fecal streptococci, a coliform and a saprophyte 0 1 1
One coliform other than E. coli 4 4 8
Two coliforms other than E. coli 6 6 12
Three coliforms other than E. coli 0 1 1
A coliform and a saprophyte 11 12 23
Two coliforms and a saprophyte 6 3 9
Saprophytes 1 2 3
No growth 0 2 2
Total 40 40 80
260 C. Mukhopadhyay et al.

Table 3 Various indicator organisms and saprophytes isolated from rural and urban water samples.
Organism No. of isolates Total

Urban Rural No. % age

Coliform bacteria (119, 70%)
Escherichia coli 07 12 19 11.2
Klebsiella spp. 26 22 48 28.2
Enterobacter spp. 11 19 30 17.6
Proteus spp. 10 04 14 8.2
Citrobacter spp. 03 05 08 4.7
Fecal streptococci (3, 1.8%)
Enterococcus spp. 03 00 03 1.8
Saprophytes (48, 28.2%)
Pseudomonas aeruginosa 17 19 36 21.2
Acinetobacter spp. 05 03 08 4.7
Serratia spp. 02 00 02 1.2
Chromobacter spp. 02 00 02 1.2
Total 86 84 170 100

antibiotics. Signicantly, among the coliform bac-
teria, ESBL production was detected in 11 (9.2%)
isolates, including E. coli (6, 31.5%), Klebsiella
spp. (3, 6.25%) and Enterobacter spp. (2, 6.6%),
from 9 water samples [Table 4]. Among the
environmental saprophytic bacilli, P. aeruginosa
was sensitive to commonly used antimicrobials,
whereas three (37.5%) isolates of Acinetobacter
spp. were resistant to more than two different
classes of antimicrobial agents.
Discussion
The examination of fecal indicator bacteria in
drinking water provides a sensitive method of qual-
ity assessment. To the best of our knowledge, this
study is the rst of its type for this region and
highlights the need for drinking water source mon-
itoring in the community for the presence of fecal
contamination along with resistance proles of the
isolates. In our study, E. coli, which provides conclu-
sive evidence of recent fecal pollution that requires
immediate attention [1,6], was detected in 23.75%
of the total samples investigated. Previous stud-
ies have reported a wide variation in the level of
E. coli contamination, ranging from 11.7% to 100%
[8,9,11,13,16]. Enterococcus spp. were detected in
three (3.75%) samples; these fecal streptococci are
more persistent than E. coli and coliform bacteria
and are highly resistant to drying; thus, they may be
valuable for detecting pollution by surface run-off
to groundwater or surface waters [6].
The total coliforms, including E. coli, were found
in a large number of well water samples (92.5%).
However, various studies around the world assessing
different water sources have revealed total col-
iform contamination rates ranging from 0% to 100%
[3,4,8,9,12]. The presence of coliform bacteria
suggests inadequate treatment or post-treatment
contamination [6]. Because a complete history of
chlorination (quantity of disinfectant used, time
since last chlorination) could not be elicited,
the inadequacy of disinfectant treatment or post-
treatment contamination with coliform bacteria
could not be assessed in the present study. Although
not always directly related to fecal contamination
or pathogens in drinking water, the coliform test
is still useful for monitoring the microbial quality
of public water supplies, largely because coliforms
are easy to detect and count in water [6]. A large
number of water sources had 10 coliforms per
100 ml, and such samples require repeat sampling
and should be further investigated for the source
of the pollution [23]. The bacterial pollution of
well water sources is mostly due to watershed ero-
sion and drainage from sewage and swamps [8].
Due to space limitations, crowding and a lack of
a proper drainage network, the septic pit sys-
tem is extensively used in this area, and seepage
from these underground pits into the nearby wells
might have contaminated the well water sources.
In our study, a large number of the samples tested
showed the presence of saprophytes, including P.
aeruginosa and Acinetobacter spp. The detection
of P. aeruginosa has been advocated as a method of
assessing the hygienic quality of drinking water [6].
A signicant observation of this study was
the nding of multi-drug resistance among both
the coliforms and saprophytes. Niemi et al.
[24] studied the distribution of the resistance
Microbial quality of well water 261

to antimicrobial drugs among fecal coliforms in

ESBL (%)

Ap, ampicillin (10 g); Ac, amoxicillinclavulanic acid (20/10 g); Cf, cefazolin (30 g); Cr, cefuroxime (30 g); Fr, ceftriaxone (30 g); Cz, ceftazidime (30 g); Pc, piperacillin
(100 g); Ci, ciprooxacin (5 g); Ct, trimethoprim-sulfamethoxazole (23.75/1.25 g); Ak, amikacin (30 g); Gm, gentamicin (10 g); Net, netilmicin (30 g); NT, not tested; ESBL,
sewage, surface waters, and sea water and found

6.25

6.6
31.5
12% of the isolates to be multi-drug resistant.

A higher rate (21%) of multi-drug resistance was
found in the present study. Vandana et al. [25]

94.4

89.5
have reported 18.8% intestinal carriage rates of
Net
100

100

100
100
100
ESBL-producing Enterobacteriaceae among newly
admitted patients. Our data collected in 2009
(unpublished) demonstrated 58.8% of all of the
97.9

89.5
Enterobacteriaceae isolates recovered from var-
100
100

100
100
100
Gm

ious infection sites to be ESBL producers. Many

of these infections could be community acquired.
The presence of multi-drug-resistant organisms in
100
100
100
100
100
100
100
Ak

drinking water sources and the above observations

suggest that well water, if consumed untreated,
can act as a source of asymptomatic colonization
95.8

93.4
89.5
92.6

and community-acquired infections with multi-drug

100
100
NT
Ct

resistant organisms. Further research should be

conducted to address this issue. In addition, the
94.4
96.7
89.5

drug resistance of one isolate can be horizontally

100

100
100
100
Ci

transferred to various other pathogenic bacterial

Antimicrobial susceptibility prole of Gram-negative bacteria isolated from water samples.

populations in water sources and in the gut, thereby

jeopardizing the existing battle against multi-drug-
97.2

resistant strains.
NT

NT
NT
NT
NT
NT
Pc

There is a need to educate the public about

the quality of their water sources and the impor-
tance of clean and healthy surroundings near water
100
NT

NT
NT
NT
NT
NT
Cz

sources and to implement measures to prevent

the contamination of water sources in the com-
munity. Boiling water is advised until disinfection
89.6

73.7
78.6

62.5
100

and retesting to conrm that the contamination

NT
90
Fr

has been eliminated. The New York State Depart-

ment of Health recommends that well owners test
their water for coliform bacteria at least once a
89.6

76.7
63.2
57.1
87.5
62.5
NT
Cr

year and recommends frequent tests in those cases

of previously documented contamination [26]. As
a pilot study, the population in this work was lim-
Antibiotics (% susceptible)

79.2

53.3
52.6
42.9
87.5

ited to a single locality, and the water quality was

NT

50
Cf

only investigated over a two-month period; there-

fore, variations in the water quality with seasonal
changes are likely to have been missed. Although
81.3

47.4
57.1
87.5
62.5
NT
Ac

60

the source of coliform contamination was not thor-

oughly investigated in this study, it is suggested that
this analysis be performed to help in implementing
26.7
36.8
42.9
87.5
37.5

control measures.
NT
Ap

extended-spectrum beta-lactamase.
0

Conclusion
Acinetobacter spp. (08)
Enterobacter spp. (30)

Citrobacter spp. (08)

Escherichia coli (19)
Klebsiella spp. (48)

It might be prudent to monitor the bacteriolog-

P. aeruginosa (36)

Proteus spp. (14)

Organisms (no.)

ical quality of well water at the source along

with resistance proles of the isolates. The impact
of multi-drug-resistant organisms in water sources
Table 4

on human health needs to be studied in greater

detail. Further studies should be performed to
determine the source of water contamination
262
and advocate proper preventive measures to the
community.
Funding: No funding sources.
Competing interests: None declared.
Ethical approval: Not required.
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