Storing Drinking-water in Copper pots Kills Contaminating Diarrhoeagenic Bacteria

Abstract

Microbially-unsafe water is still a major concern in most developing countries. Although many water-purification methods exist, these are expensive and beyond the reach of many people, especially in rural areas. Ayurveda recommends the use of copper for storing drinking-water. Therefore, the objective of this study was to evaluate the effect of copper pot on microbially-contaminated drinking-water. The antibacterial effect of copper pot against important diarrhoeagenic bacteria, including Vibrio cholerae O1, Shigella flexneri 2a, enterotoxigenic Escherichia coli, enteropathogenic E. coli, Salmonella enterica Typhi, and Salmonella Paratyphi is reported. When drinking-water (pH 7.83 +/- 0.4; source: ground) was contaminated with 500 CFU/mL of the above bacteria and stored in copper pots for 16 hours at room temperature, no bacteria could be recovered on the culture medium. Recovery failed even after resuscitation in enrichment broth, followed by plating on selective media, indicating loss of culturability. This is the first report on the effect of copper on S. flexneri 2a, enteropathogenic E. coli, and Salmonella Paratyphi. After 16 hours, there was a slight increase in the pH of water from 7.83 to 7.93 in the copper pots while the other physicochemical parameters remained unchanged. Copper content (177 +/- 16 ppb) in water stored in copper pots was well within the permissible limits of the World Health Organization. Copper holds promise as a point-of-use solution for microbial purification of drinking-water, especially in developing countries.

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©INTERNATIONAL CENTRE FOR DIARRHOEAL
DISEASE RESEARCH, BANGLADESH
Correspondence and reprint requests should be
addressed to:
Dr. Padma Venkatasubramanian
Centre for Pharmacognosy, Pharmaceutics and
Pharmacology
Institute of Ayurveda and Integrative Medicine
74/2 Jarakabande Kaval, Yelahanka via Attur
Bangalore 560 0106, Karnataka
India
Email: padma.venkat@frlht.org OR
padmavenkatl@rediffmail.com
Fax: +91 080 28567926
Storing Drinking-water in Copper pots Kills
Contaminating Diarrhoeagenic Bacteria
V.B. Preethi Sudha1, Sheeba Ganesan1, G.P. Pazhani2, T. Ramamurthy2,
G.B. Nair2, and Padma Venkatasubramanian1
1Centre for Pharmacognosy, Pharmaceutics and Pharmacology, Institute of Ayurveda and Integrative Medicine (formerly Foundation
for Revitalisation of Local Health Traditions), 74/2 Jarakabande Kaval, Yelahanka via Attur, Bangalore 560 0106, Karnataka, India and
2National Institute of Cholera and Enteric Diseases, P-33, CIT Road, Scheme XM, Beliaghata, Kolkata 700 010, West Bengal, India
ABSTRACT
Microbially-unsafe water is still a major concern in most developing countries. Although many water-puri-
fication methods exist, these are expensive and beyond the reach of many people, especially in rural areas.
Ayurveda recommends the use of copper for storing drinking-water. Therefore, the objective of this study
was to evaluate the effect of copper pot on microbially-contaminated drinking-water. The antibacterial
effect of copper pot against important diarrhoeagenic bacteria, including Vibrio cholerae O1, Shigella flexneri
2a, enterotoxigenic Escherichia coli, enteropathogenic E. coli, Salmonella enterica Typhi, and Salmonella Pa-
ratyphi is reported. When drinking-water (pH 7.83±0.4; source: ground) was contaminated with 500
CFU/mL of the above bacteria and stored in copper pots for 16 hours at room temperature, no bacteria
could be recovered on the culture medium. Recovery failed even after resuscitation in enrichment broth,
followed by plating on selective media, indicating loss of culturability. This is the first report on the effect
of copper on S. flexneri 2a, enteropathogenic E. coli, and Salmonella Paratyphi. After 16 hours, there was a
slight increase in the pH of water from 7.83 to 7.93 in the copper pots while the other physicochemical
parameters remained unchanged. Copper content (177±16 ppb) in water stored in copper pots was well
within the permissible limits of the World Health Organization. Copper holds promise as a point-of-use
solution for microbial purification of drinking-water, especially in developing countries.
Key words: Bacteria; Copper; Diarrhoea; Drinking-water; Vibrio cholerae; India
J HEALTH POPUL NUTR 2012 Mar;30(1):17-21
ISSN 1606-0997 | $ 5.00+0.20
INTRODUCTION
Providing safe drinking-water to the majority of
the world’s population, especially to those in devel-
oping countries, is still a major problem. Approxi-
mately a billion people lack access to safe drink-
ing-water (1). Water and food contaminated with
bacteria, viruses, and protozoa cause infectious
diarrhoea. Diarrhoea is one of the leading causes
of mortality and morbidity, especially in children
of developing countries (2) and claims two million
lives each year (3). The major aetiological agents
that account for over a million diarrhoeal deaths
per year, particularly in developing countries, are
enterotoxigenic Escherichia coli (ETEC), rotavirus,
Vibrio cholerae, and species of Shigella, which are
spread through contaminated water and food or
from person to person (4). In India, many states
still have outbreaks of cholera. During 1996-2007,
at least 222,038 individuals were affected by chole-
ra (5). Shigellosis, also known as acute bacillary
dysentery, is associated with complications, such as
haemolytic-uraemic syndrome which can be fatal
(6). Shigella flexneri causes approximately 10% of all
diarrhoeal episodes among children aged less than
five years (7). Infection with ETEC is associated
with traveller’s diarrhoea, and the rate of infec-
tion is higher in India compared to other develop-
ing countries (8). Among the viruses, rotaviruses
are the most common cause of diarrhoea in infants
and children. In Asia, rotaviruses are responsi-
ble for 45% of hospitalizations for severe infantile
diarrhoea (9). Microbial quality, though only one
of the parameters of safe drinking-water, is a major
problem and is a cause of epidemics in developing

Sudha VBP et al.
JHPN
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Copper kills diarrhoeagenic bacteria
countries. The existing community interventions
to provide safe drinking-water to the people have
many shortcomings, and studies have shown that
point-of-use (PoU) household interventions con-
tribute to 30-40% reduction in diarrhoeal diseases
(10). Moreover, in countries such as India where
only 28% of households have piped water (5), PoU
interventions are a sustainable way to providing
safe drinking-water.
Storing water in copper and silver pots finds men-
tion in ancient texts of Ayurveda for purification of
water (11). Our previous study provided laboratory
evidence of the antibacterial activity of copper pot
in distilled water (12). We had also reported the
benefit of using a copper-based device, contrived
by us, which was as effective as the pot but at
a fraction of the cost (12). Since distilled water is
slightly acidic (pH 6.7±0.05) which might en-
hance copper leaching, we have demonstrated the
effect of copper pot in regular drinking-water (pH
7.83±0.4) against important bacterial pathogenic
strains that cause diarrhoea.
MATERIALS AND METHODS
Bacterial strains
V. cholerae O1 IDH 02474 (VC), S. flexneri type 2a
IDH 02196 (SF), Salmonella enterica Typhi 500865
(SET), and enterotoxigenic E. coli (LT+ST) IDH
01254 (ETEC) were obtained from the National Ins-
titute of Cholera and Enteric Diseases (NICED),
Kolkata, India. S. Paratyphi A B/05 (SPT) was pro-
cured from the St. Johns Medical College, Banga-
lore, India and confirmed at NICED. Enteropatho-
genic E. coli E 2347 (EPEC) was obtained from the
Christian Medical College (CMC), Vellore, India.
Preparation of bacterial cultures
Cultures from the nutrient agar culture-stab were
streaked onto selective media, including eosin
methylene blue (EMB) agar medium (HIMEDIA,
Mumbai, India) for E. coli species, xylose lysine dex-
trose (XLD) medium (HIMEDIA) for Salmonella spe-
cies, Henktoen enteric agar (HEA) medium (Difco,
USA) for Shigella, and thiosulphate-citrate bile-salts
sucrose (TCBS) medium (HIMEDIA) for V. cholerae,
and were incubated at 37 0C for 16-18 hours in a
bacteriological incubator (IN 18 DF, Servewell In-
struments Private Limited, Bangalore, India). After
incubation, a single colony was picked and inocu-
lated into 2 mL of Luria Bertani broth (Difco) and
incubated for 16-18 hours in a bacteriological incu-
bator at 37 oC. This overnight culture was serially
diluted in normal saline (NaCl, 0.85%) for inocula-
tion in water.
Antibacterial activity of copper pot on drinking-
water inoculated with enteric pathogens
The experiment procedure followed was essen-
tially as per Sudha et al. (12). Copper pots of 2-L
capacity (test) purchased from local vendors were
thoroughly cleaned and autoclaved each time be-
fore use. Presterilized 1-L glass bottles (Schott Du-
ran, Mainz, Germany) acted as controls.
Water was collected from the tap (groundwater,
pumped to the overhead tank) from the Microbiolo-
gy Unit of FRLHT (Foundation for Revitalisation of
Local Health Traditions), Bangalore and was auto-
claved. The sterilized water was inoculated to
~500 colony-farming unit (CFU)/mL with serially-
diluted overnight culture of the diarrhoeagenic
bacteria. The same was enumerated by spread plate
method on nutrient agar (HIMEDIA). Two litre
of inoculated water was poured into copper pots
(2x2L) and one litre into each of the two prest-
erilized Schott Duran bottles. After incubation at
room temperature (26±2 oC) for 16 hours, 100 μL
of samples was withdrawn, after mixing, from each
container and plated on nutrient agar for the enu-
meration of bacteria. Resuscitation of sublethal-
ly-damaged cells was monitored by enrichment
method (13). Three mL of test or control water
sample was mixed with an equal volume of double-
strength peptone water (enrichment medium) and
incubated for 24 hours at 37 oC. After incubation,
the medium was observed for turbidity, and also a
loopful of the enriched culture was streaked onto
respective selective media as mentioned earlier and
observed for growth after incubation for 24 hours
at 37 oC. All experiments were conducted three
times with duplicates maintained each time.
Analysis of physical and chemical parameters
of water
Tests and controls of the inoculated water were
assessed before and after incubation for physi-
cochemical properties, including pH, turbidity,
total dissolved solids (TDS), alkalinity, hardness,
contents of chlorides and sulphates as per proto-
cols of the Bureau of Indian Standards (14). The pH
was measured using a pH meter (DI 707; Digisun
Electronics, Hyderabad, India). Copper content was
estimated using Spectroquant (Merck, Darmstadt,
Germany), a commercially-available, ready-to-use
kit, as described in Sudha et al. (12).
RESULTS
Antibacterial activity of copper pot on drink-
ing-water inoculated with enteric pathogens
VC, SF, ETEC, EPEC, SET, and SPT inoculated
into water could not be recovered on the specific

Sudha VBP et al.
Copper kills diarrhoeagenic bacteria
Volume 30 | Number 1 | March 2012 19
Table 1. Effect of overnight storage of tap-water inoculated with diarrhoeagenic bacteria in copper pots
and glass bottles
Bacteria
inoculated
Before
incubation
Copper pots Glass bottles
After incubation After incubation
Dose
(CFU/mL)
Bacterial count
(CFU/mL)
Enrichment
culture
Bacterial count
(CFU/mL)
Enrichment
culture
V. cholerae O1 IDH
2474 506±11 No growth Not detected 516±11 Detected
S. flexneri 2a IDH
02196 533±28 No growth Not detected 530±26 Detected
ETEC IDH 01254 513±23 No growth Not detected 866±83 Detected
EPEC E2347 506±11 No growth Not detected 600±10 Detected
S. enterica Typhi
500865 170±53 No growth Not detected 109±66 Detected
S. Paratyphi A 453±109 No growth Not detected 361±67 Detected
CFU=Colony-forming unit; EPEC=Enteropathogenic Escherichia coli; ETEC=Enterotoxigenic Escherichia
coli
growth medium as mentioned in methods (Ta-
ble 1). In the control glass bottles, on the other
hand, the number of bacteria inoculated either
remained the same or slightly increased (Table 1).
After incubation in the enrichment broth, there
was no visible turbidity in the test samples, and
no bacteria could be recovered when the enriched
cultures were streaked onto selective media. With
controls, turbidity in enrichment medium and
subsequent growth of bacteria on selective me-
dium were observed (Table 1), where VC exhib-
ited as typical yellow colony on TCBS medium,
SF as typical small green colonies on HEA medi-
um, and Salmonella species as pink colonies with/
without black centre on XLD medium whereas
ETEC and EPEC exhibited typical metallic sheen
colonies on EMB agar medium. This indicates
that the bacteria in the test samples were either
completely killed or had lost their culturabili-
ty on media.
Physical and chemical parameters
The level of copper that had leached into the test
samples was 177±16 ppb which was well within
the WHO limit of 2000 ppb (Table 2). TDS, alkalin-
ity, hardness, contents of chlorides and sulphates
remained the same before and after incubation in
both test and control samples, except that the pH
had slightly increased from 7.83±0.4 to 7.93±0.3 in
the test samples (copper pot) after incubation for
16 hours (Table 2).
DISCUSSION
None of the test pathogens was recovered from
drinking-water stored in copper pots even after
enrichment culture. This is the first report on the
antibacterial activity of copper against pathogenic
strains of SF, EPEC, and SPT. Copper pot is as active in
regular drinking-water (pH 7.83±0.4) as that report-
ed by us earlier (12) in distilled water (pH 6.7±0.05),
and the level of copper leached in the former is far
less (177±16 ppb) than that in distilled water (~420
ppb). Other studies have shown that copper ves-
sel is lethal to E. coli in water at different pH and
temperature conditions, with the fastest inacti-
vation occurring as the pH shifts away from neu-
trality and 35 oC (15). Copper has also been shown
to act, to a greater or lesser extent, on E. coli in the
presence of organic and inorganic constituents in
water (16). In laboratory experiments, copper has
been shown to kill meticillin-resistant Staphylococ-
cus aureus (17), Campylobacter jejuni, and S. enterica
(18). Findings of these studies suggest that copper
can act on a range of organisms under different
conditions. It is still important to test the effect of
copper on various sources of drinking-water under
different field conditions. Safety of leached copper
does not appear to be an issue since studies have
shown that the current WHO guideline of 2 mg
Cu/L is safe (19,20), and the levels leached in the
study were ~1/20th of the permissible limits. It has
been observed in the present study that the other
physicochemical parameters of drinking-water re-
main unchanged after copper intervention, which
makes them amenable for public use.
We observed that the unrecovered bacteria in the
test samples did not get resuscitated even after
enrichment and plating on selective media. This
indicates that they have lost culturability on non-
selective medium and on enrichment and selective
media. However, we still need to confirm whether

Sudha VBP et al.
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20
Copper kills diarrhoeagenic bacteria
Table 2. Physicochemical quality of tap-water before and after incubation in copper pot and in
glass bottles
Parameter Permissible limit
(BIS/WHO*)
Before
incubation
After incubation
Test Control
Alkalinity (mg/L) 600 25 25 25
Hardness (mg/L) 600 280 280 280
Turbidity (NTU) 10 0.47 0.47 0.47
TDS (mg/L) 2,000 700±49.5 655±35.4 690±14
Chlorides (mg/L) 1,000 35.45 35.45 35.45
Sulphates (mg/L) 400 86.5 86.5 86.5
pH 8.5-9.0 7.83±0.4 7.93±0.3 7.83±0.4
Copper content (mg/L)*2 <DL 0.1 0.177±0.016 <DL
*Detectable limit=0.02 mg/L; BIS=Bureau of Indian Standards; DL=Detecting limit; NTU=Nephelometric
turbidity unit; TDS=Total dissolved solids; WHO=World Health Organization
they have transformed into the viable but non-
culturable (VBNC) state. VBNC is a state in bacte-
ria where the cells do not grow onto routinely-
employed media but are still viable (21). VBNC
bacteria have been studied using several methods,
including alteration in temperature (22), use of en-
richment medium (23), changes in the growth
medium, using chick embryo yolk sack, passage
through rabbit illeal loop (13), or co-culturing
with eukaryotic cell cultures (21). VBNC bacteria
have been observed using viable stains (22) and
microscopy (21,24,25).
Studies have shown that copper surfaces completely
kill bacteria. E. coli inoculated on to copper coupons
were completely killed. The studies concluded that
the copper ions brought about complete killing of
bacteria by membrane damage (26). However, the
mechanism of action of copper on bacteria is not
completely understood.
Although studies have shown the merits of copper
surfaces for their use in improving public hygiene
in healthcare facilities, the potential use of copper
for the purification of drinking-water, especially in
developing countries, has not been widely stud-
ied. Therefore, results of our study indicate that
copper holds potential to provide microbially-safe
drinking-water to the rural masses in develop-
ing countries. The use of copper pots in Indian
households is common and is, therefore, likely to
be socially accepted by the people. Its functioning
is not dependent on fuel, electricity, replaceable
filters, intensity of sunlight, etc. to operate or
maintain it; it is simply a passive storage of water.
This takes into account the conditions prevailing
in rural villages and the urban slums of developing
countries. The health benefit that can be achieved
by using copper pot as a PoU water-purification de-
vice will far outweigh the cost of the pot, if divided
over the members in a rural family, especially as it
will be a one-time investment with no recurring
costs. However, it is important to challenge its use
under real-life conditions in the dynamics of the
target households in developing countries to fully
understand the limitations.
ACKNOWLEDGEMENTS
The study was supported by the ETC CAPTURED
Programme, The Netherlands (Grant No. DGIS/D).
The authors thank Mr. Darshan Shankar, Advisor,
IAIM, for his constant encouragement.
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