Drinking water stored in copper vessel - reveals antibacterial activity

Abstract

Introduction and Aim: Recently About 2.2 million diarrheal deaths per year especially in developing countries, in which 1.4 million deaths among children under age five. Safe drinking water, especially in developing countries, is still a major problem. The present study was designed to explore the antibacterial effect of copper vessel stored with drinking water inoculated with diarrhea-causing bacterial strains enterotoxigenic Escherichia coli (ETEC), Vibrio cholera and Shigella flexneri. Materials and Methods: The bacterial strains isolated from fecal sample, identified by colony morphology and biochemical test. About 500 Colony Forming Unit (CFU/mL) of the bacterial strains of E. coli, V. cholera and S. flexneri were inoculated separately in drinking water stored in the copper vessel and non- copper vessel for 12 hrs. After incubation 100 μL of samples was taken from each copper vessel and non-copper vessel container and spread on nutrient agar for the enumeration of bacteria colonies. After 24hrs of incubation at 37°C, dishes were observed for visible bacterial colonies using colony counter. Results: The bacterial strains E. coli (ETEC) (532 CFU), V. cholera (502 CFU) and S. flexneri (512 CFU) was inoculated in copper vessel observed no growth on the selective media and when the bacterial strains inoculated in the non-copper vessel observed growth upon inoculated in selective media E. coli (ETEC) (624 CFU), V. cholera (328 CFU) and S. flexneri (483 CFU). Conclusion: The antibacterial activity may be due to the oligodynamic effect of copper which destroy the cell wall and cell membrane results in membrane damage. The present study recommends the use of cost-effective copper vessels to store drinking water especially in rural areas for protection from water-borne infection. © 2018 Indian Association of Biomedical Scientists. All rights reserved.

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Biomedicine: 2016; 36(3): 96-071
Drinking Water Stored in Copper Vessel - Reveals Antibacterial Activity
Jayalakshmi G1, Swathi S1, Naveen Kumar C1, Srikumar R2, Vijayakumar R3
Manikandan S4, Manoharan A5 and Chidambaram R2
1Department of Microbiology, 2Centre for Research, 3Department of Physiology, 5Department of Pathology,
Sri Lakshmi Narayana Institute of Medical Sciences, Aliated to Bharath University, Pondicherry,
4Department of Physiology, Tagore Medical College & Hospital, Melakkottaiyur Post, Chennai.
(Received: Oct 2017 Revised: Dec 2017 Accepted: Feb 2018 )
Corresponding Author
Srikumar R. E-mail: rsrikumar_2003@yahoo.co.in
ABSTRACT
Introduction and Aim: Recently About 2.2 million diarrheal deaths per year especially in developing coun-
tries, in which 1.4 million deaths among children under age ve. Safe drinking water, especially in developing
countries, is still a major problem. The present study was designed to explore the antibacterial eect of copper
vessel stored with drinking water inoculated with diarrhea-causing bacterial strains enterotoxigenic Escherich-
ia coli (ETEC), Vibrio cholera and Shigella exneri.
Materials and Methods: The bacterial strains isolated from fecal sample, identied by colony morphology
and biochemical test. About 500 Colony Forming Unit (CFU/mL) of the bacterial strains of E. coli, V. cholera
and S. exneri were inoculated separately in drinking water stored in the copper vessel and non- copper vessel
for 12 hrs. After incubation 100 μL of samples was taken from each copper vessel and non-copper vessel con-
tainer and spread on nutrient agar for the enumeration of bacteria colonies. After 24hrs of incubation at 37°C,
dishes were observed for visible bacterial colonies using colony counter.
Results: The bacterial strains E. coli (ETEC) (532 CFU), V. cholera (502 CFU) and S. exneri (512 CFU) was
inoculated in copper vessel observed no growth on the selective media and when the bacterial strains inoculat-
ed in the non-copper vessel observed growth upon inoculated in selective media E. coli (ETEC) (624 CFU),
V. cholera (328 CFU) and S. exneri (483 CFU).
Conclusion: The antibacterial activity may be due to the oligodynamic eect of copper which destroy the cell
wall and cell membrane results in membrane damage. The present study recommends the use of cost-eective
copper vessels to store drinking water especially in rural areas for protection from water-borne infection.
Key Words: Diarrheal; Copper vessel, Drinking water
INTRODUCTION
The 70% of the World is covered by water of
which only 2.5% is of fresh water, which 1%
of the freshwater is accessible easily, remain-
ing trapped in glaciers and snowelds and from the
1% of fresh water only 0.3-0.5% is available for
drinking purpose. As the human population enhanc-
es, day by day and daily consumption of fresh water
also increased several folds.
Water plays an essential dual role - one is vital for
life, as more than 60% of human body is composed
of water and sustains the natural environment, con-
tributes to the development of economic, health, so-
cial, recreational and cultural activities. On the other
hand, it plays a vital role in the transmission of wa-
ter-borne infectious diseases. It is estimated to cause
about 2.2 million diarrhea deaths per year especially
in developing countries in which 1.4 million deaths
among children under age ve (1). Worldwide, an
economic burden for a water-borne disease is about
12 billion US dollars per year (2). Waterborne in-
fections are caused by ingestion, airborne or contact
with contaminated water by a variety of infectious
agents which includes bacteria, viruses, protozoa
and helminths (3). In addition, ood accelerates the
risks of outbreaks of waterborne diseases. About
1415 species of microorganisms have been reported
to be pathogenic, among which approximately 348
are water-borne, causing 115 infectious diseases (4).
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From 1991 to 2008, about 1,428 water-borne out-
breaks were reported (5).
The major bacterial agents that account for millions
of diarrhoeal deaths particularly in developing coun-
tries, are enterotoxigenic Escherichia coli (ETEC),
Vibrio cholerae, and species of Shigella, which
spread through contaminated water, food or from
person to person. Shigella exneri known as acute
bacillary dysentery causes approximately 10% of all
diarrhoeal episodes among children aged less than
ve years (6). Infection with ETEC is associated
with traveler’s diarrhea, and the rate of infection is
higher in India compared to other developing coun-
tries (7).
Currently, about 780 million people do not have
access to a puried drinking water, and 2.5 billion
people lack access to improve sanitation worldwide
especially in the developing countries (8). It is es-
timated that about 3.2% of deaths globally are at-
tributable to consume unsafe water caused by poor
sanitation and hygiene (9). The WHO recommends
that improving water quality may reduce the global
diseases burden by approximately 4%. Thus, there is
an urgent need to move all possible eorts to min-
imize the water-borne infection. Though detection
methods play an essential role in monitoring water
quality, surveillance, and quantitative microbial risk
assessment but providing safe drinking water glob-
ally especially in developing countries like India is
still a challenging one.
The household boiling water for disinfection is in
danger of leaching harmful chemicals from the plas-
tic bottles and container, also prone to recontamina-
tion during handling and storage. At present three
main water treatment methods are available includes
distillation, reverse Osmosis and solid block acti-
vated carbon, apart UV, Ozone, activated alumina,
sediment lters, ion exchange, granular activated
carbon, and boiling are also used but combined with
other methods for eective outcomes. Many of the
currently available water purication systems are
more expensive, required regular maintenance, elec-
tricity and beyond the reach of the rural population
in countries such as India is questionable. Even in-
expensive Candle lters (with diatomaceous earth)
require regular cleaning and replacement are usually
ignored by users.
In most of the rural places at India, people usually
collect drinking water from the lake, ponds, wells,
running streams, municipal pipes, from stored water
tanks and water, may become contaminated at any
point between collection, carrying and storage be-
fore use. The use of copper by human civilizations
dates behind to between the 5th and 6th millennia
B.C. It was the 1st metal used, presumably because
it could be found in a native, metallic form which did
not demand to smelt. The Indian Ayurveda describes
storing water in a copper vessel overnight and drink-
ing it in the morning had many health benets. The
present study was designed to explore the antibac-
terial eect of copper vessel container by storing
the drinking water inoculated with diarrhea causing
bacterial strains enterotoxigenic E. coli (ETEC), V.
cholera and S. exneri.
MATERIALS AND METHODS
Isolation and Identication of Bacterial Strains
Bacterial strains enterotoxigenic E. coli (ETEC),
V. cholera and S. exneri were isolated in the fae-
cal sample. Bacterial strains enterotoxigenic E. coli
(ETEC), V.cholera and S. exneri were isolated in
the stool samples by culturing on Mac conkey agar,
thiosulphate-citrate-bile salt sucrose agar (TCBS)
and Salmonella shigella agar respectively. Initially
colony morphological identication was Mac con-
key plates showed pure lactose fermenting, trans-
lucent, glossy, smooth colonies and were identied
as E. coli. On TCBS agar V. cholera produce large,
attened yellow colour colonies with opaque centers
due to fermentation of sucrose in the medium. S.
exneri is non lactose fermenter appear as a trans-
parent colorless colonies. Biochemical test showed
Indole and Methyl Red (MR) showed Positive and
negative for Voges-Proskaure (VP) and citrate for E.
coli. For identication of V. cholera Catalase and ox-
idase test showed positive, urease test showed nega-
tive, indole, citrate and MR test showed positive and
Negative for VP test. For identication of S. exneri
showed Catalase, MR and nitrate reduction showed
positive and negative for Citrate, Oxidase, and VP
test. After morphological and biochemical identi-
cation single colonies of E. coli (ETEC), V. cholera
and S. exneri were isolated, inoculated in sterile
Luria Bertani broth as a starter culture and incubated
at 370Cfor 12- 16 hrs, followed by serial dilution
with normal physiological saline.
Serial Dilution Method
A pure culture may be obtained by serially diluting
(tenfold-1 in 10 dilutions-1:10, 1:100 or 1/10, 1/100,
1/1000 or 10-1, 10-2, 10-3) the sample with sterile
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water or saline to the point of extinction in numbers
of cells and transferred on an agar plate. In serial
dilution technique sample has been diluted serially.
It is assumed that each viable bacterium in the orig-
inal sample can produce one discrete colony (col-
ony forming unit or CFU) and thus, the number of
colonies represents the number of bacteria that can
grow under the conditions. Petridish with between
30 and 300 colonies are ideal for completing a stan-
dard plate count.
Spread Plate Method
An aliquot of the serially diluted sample (0.1ml) is
placed onto the agar surface in a petri dish and is
spread uniformly with a sterile, bent ‘L’ shaped glass
rod until the surface becomes dry and the spreader
begins to stick to the surface. Petri dishes are turned
upside down to avoid condense on the agar and in-
cubated at 37oC for 24 hours and after 24 hours of
incubation the Petri dishes are examined for visible
the colony formation. Above said procedure repeat-
ed in triplicate and average mean of the three was
calculated.
Inoculation of bacterial culture in sterilized wa-
ter stored in copper and non-copper vessel
Drinking water (2 liters) was sterilization at 120°C
for 15-20 minutes and transferred to sterile copper
vessel and non-copper vessel. Then sterilized water
was inoculated with 500 CFU/mL of test bacteri-
al strain. The CFU was carried by serially dilution
and spread plate method (for each bacterial culture
separate copper / non-copper containers were used).
Before and after inoculation bacterial population
was enumerated by spread plate method. Then the
copper vessel and non-copper vessel’s with bacterial
cultures were incubate at room temperature (28±2
°C) for 12hours. After incubation 100 μL of samples
was taken from each copper vessel and non-copper
vessel container and spread on nutrient agar for the
enumeration of bacteria colonies. After 24hrs of in-
cubation at 37°C, dishes were observed for bacterial
colonies using colony counter.
RESULTS
The bacterial strains E. coli (ETEC) (532 CFU), V.
cholera (502 CFU) and S. exneri (512 CFU) was
inoculated in copper vessel showed no growth on the
selective media and when the bacterial strains inoc-
ulated in the non-copper vessel showed growth upon
inoculated in selective media E. coli (ETEC) (624
CFU), V. cholera (328 CFU) and S. exneri (483
CFU) shown in Table No. 1.
Table 1: Eect of copper vessel container on wa-
ter sample with diarrhoeagnic bacteria stored for
12 hrs
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Sl.
No
Bacterial
sample
Before incubation
number of CFU*
Aer
incubation
number
of CFU
for sample
taken cop-
per vessels
container
Aer
incubation
number
of CFU
for sam-
ple taken
non-copper
vessels
container
1Esche-
richia coli
(ETEC),
532±24 0624±35
2Vibrio
cholera
502±12 0328±24
3Shigella
exneri
512±11 0483±56
* Number of colony forming unit (CFU)
DISCUSSION
Water is essential for maintaining life on Earth, but
can also serve as a media for many pathogenic or-
ganisms. The present study was designed to provide
safe water to people especially at the rural places
where the water purier cannot reach since more
expensive, required regular maintenance which is
beyond for the rural population. The use of copper
by human civilizations dates back to between the
5th and 6th millennia B.C. Copper vessel is merely
passive storage of water, and it will be a one-time
investment with no recurring costs for further main-
tenance. It is suitable for developing countries like
India where there is a frequent intermittent supply
of drinking water, necessitating storage of drinking
water for days. In such conditions, copper vessels
can be introduced during storage of drinking water.
In the present study, the result showed, no growth
was observed after 12hrs of incubation of bacterial
strains stored in a copper vessel and when the water
stored in non-copper vessel showed the presence of
colony forming unit indicates its viability (Table No.
1).
When the water is stored in a copper vessel over-
night a detectable amount of copper ions gets dis-
solved into the water called Oligodynamic eect.
The dissolved copper result in structural and mem-
brane damage, by observing to inactivate bacteria by
destroying their cell wall and cell membrane (10,11).
Damage to the membrane leads to leakage of potas-
sium or glutamate through the outer membrane of
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bacteria results in osmotic shock. In addition, copper
ions binding to proteins that cause oxidative stress
by generating hydrogen peroxide and damaging
DNA may result in genotoxicity (11).
As copper vessel are not very common today since
the costly and easy availability of plastic and stain-
less steel containers with less cost. The present study
recommends to use a copper vessel to store drinking
water since Copper vessel is easy to use, requires
no electricity/battery and does not need any mainte-
nance and replacements like other commercial water
puriers, making it ideal for situation prevailing in
developing countries like India especially in rural ar-
eas. This study also recommends that canned drink-
ing water supply companies can use copper coated
containers for processing the water and also to use
copper coated pipelines.
CONCLUSION
The antibacterial properties of water stored in cop-
per vessel inoculated with bacterial strains were
rmly established. The result showed no bacterial
colony counts were enumerated which implies that
storage of water in the copper vessel is a promising
additional tool alongside other hygienic measures
to curb the number and severity of waterborne di-
arrhoeal infections. At this point, additional studies
would be recommended to help in determining the
most cost-eective way to give maximal protection
for other water-borne diseases.
Conicts of Interest
Author’s declared that no Conict of Interest
REFERENCES
1. World Health Organization (WHO) Water Sani-
tation and Health. 2015. [(assessed on 17 Febru-
ary 2015)]. Available online: http: // www .who
.int/water_ sanitation_ health/diseases.
2. Alhamlan Al-Qahtani, F.S., and Al-Ahdal, A.A.
Recommended advanced techniques for water-
borne pathogen detection in developing coun-
tries. J infect develop count. 2015; 9: 128-135.
3. Ingerson-Mahar, M., and Reid, A. Microbes in
Pipes: The Microbiology of the Water Distribu-
tion System A Report on an American Academy
of Microbiology Colloquium. ASM Acad; Boul-
der, CO, USA. 2012; 26.
4. Taylor, L.H., Latham, S.M., and Woolhouse,
M.E. Risk factors for human disease emergence.
Philos Transact Royal Soc B: Biol Sci. 2001;
356: 983-989.
5. Kun, Y., Jerey, L., Doug, A., Bo, L., Shum,
C.K., and Song, L. Global Distribution of Out-
breaks of Water-Associated Infectious Diseases.
PLOS Neglected Tropical Diseases. 2012; 6:
e1483.
6. Niyogi, S.K. Shigellosis. J Microbiol. 2005; 43:
133-143.
7. Shah, N., Dupont, H.L., and Ramsey, J.D. Glob-
al etiology of travelers’ diarrhea: systematic re-
view from 1973 to the present. Am J Trop Med
Hyg. 2009; 80: 609-614,
8. Centers for Disease Control and Prevention
(CDC) Global Water, Sanitation, and Hy-
giene(WASH) Availableonline: http://www.cdc.
gov/healthywater/global/ [(assesed on 17 Febru-
ary 2015)]
9. World Health Organization (WHO) “Progress on
Sanitation and Drinking-Water: Update Report”,
World Health Organization (WHO); Geneva,
Switzerland, 2010.
10. Warnes, S.L., Caves, V., and Keevil, C.W. Mech-
anism of copper surface toxicity in Escherichia
coli O157:H7 and Salmonella involves immedi-
ate membrane depolarization followed by slower
rate of DNA destruction which diers from that
observed for Gram-positive bacteria. Environ
Microbiol. 2012; 14: 1730-1743.
11. Hong, R., Kang, T.Y., Michels C.A., and Gadu-
ra, N. Membrane lipid peroxidation in copper al-
loy-mediated contact killing of Escherichia coli.
Appl Environ Microbiol. 2012; 78: 1776-1784.
12. Warnes, S.L., Green, S.M., Michels, H.T. and
Keevil, C.W. Biocidal ecacy of copper alloys
against pathogenic enterococci involves degra-
dation of genomic and plasmid DNA. Appl En-
viron Microbiol. 2010; 76: 5390-5540.
Tamilselvan et al.: Eect of Menstrual Cycle..................................Mucociliary Clearance
FOR PROOFREADING