For controlling of bacterial contaminants in
drinking water sources, there is a need for some
new techniques to removing of pathogenic bacteria from drinking sources using
an innovative method to achieve the target of water purification.  Therefore, this study was aimed to evaluate
the biocidal activity of calcium hypochlorite Ca(OCl)2, silver
nanoparticles (AgNPs) and its loaded forms against isolated bacteria from
drinking water supplies (tap and hand pump water) then implemented a field trial
to evaluate a biocidal filter paper containing nanocomposite against indicator
coliforms bacteria. One hundred water samples were collected from the main
source and water trough used for cattle drinking then examined for isolation
and identification of pathogenic bacteria. Sixty strains of isolated bacteria
were chosen to evaluate its sensitivity to Ca(OCl)2, AgNPs and AgNPs
loaded Ca(OCl)2 using broth macro dilution method. After that, a field
trial was applied through examination of different water samples collected from
both water supplies pre and post-treatment using a filter paper impregnated in AgNPs/Ca(OCl)2
in order to evaluate its biocidal effect on indicator coliforms bacteria. Results,
AgNPs/Ca(OCl)2 nanocomposite at a concentration of 0.002 mg/L showed
the lethal effect on  E. Coli, S.
aureus and K. Pneumonae (100% each) after 60 minutes of the
exposure time compared to the lowest concentration of nanocomposite.
Furthermore, AgNPs/Ca(OCl)2
filter paper exhibited biocidal effect
(100%) toward total viable bacterial count (TVC) CFU/ mL, total coliforms (TCC)
and fecal coliforms bacteria (FCC) CFU/100 mL compared to water samples pre-treatment
process. Conclusions, testing of Ca(OCl)2
disinfectant against isolated bacteria exhibited the existing of bacterial
resistance profile. Therefore, a new trend that towards to enhancement of the biocidal
effect of tested disinfectant using silver nanoparticles was improved that AgNPs/Ca(OCl)2 disinfectant had a biocidal effect (100%) against isolated bacteria in addition to inactivation
of coliforms bacteria during
percolation through the bactericidal filter paper. 

Keywords: calcium hypochlorite. Water purification. Bactericidal filter paper. AgNPs.         

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advances in the field of water management practices, waterborne pathogen
remains one of the main challenges and threats to both human and animal health (CDC,
2013). E. coli is the main recognized indicator microorganism of fecal
contamination in water sources (Health Canada, 2012; Odonkor and Ampofo,
2013). For instance, coliforms and E. coli are thermos-tolerant
bacterial groups that indicate the presence of fecal matter (Savichtcheva and
Okabe, 2006).  To ensure water safety
and protect both the public and animal health, the disinfection process is
considered the main step whereas many scientists have been exploring various
methods to improve the disinfectants performance through disinfection
strategies for water treatment (Xu Yang, 2016).

the different methods of disinfection include chemical, physical and
photochemical methods are essential for the treatment of contaminated water.
Chemical disinfectant such as calcium
hypochlorite is based on the oxidation of the chemical itself and determines
the extent of damage towards the bacterial cell walls (Randtke, 2010). As
chlorine is still popular disinfectant due to its ease of application, the low
cost (Van Haute et al., 2013) and it can react with the cell membrane, cause of cell lysis and
microbial death (Bitton, 2011)


Using of
Nano-silver as the antimicrobial agent is attractive for many reasons: it
hasn’t unpleasant effects on the odor, taste, and color of drinking water.
Furthermore, its activity against different types of microorganisms was obvious
(Kim et al. 2007); the occurrence of bacterial resistance to silver is
dif?cult and almost no adverse impacts on both human beings and animal health
if digested in low amount. There are numerous applications of silver and silver
compounds that using as the antimicrobial agents, portable water ?lters, and medical
devices. Sondi and Salopek-Sondi (2004) ?rst studied nanosilver as the antimicrobial
agent against E. coli bacteria. In this study nanosilver was synthesized
through reduction of silver nitrate using ascorbic acid (Sondi et al., 2003)
Furthermore, 70% of inactivated E. coli as Gram-negative bacteria
was recorded at a dose of 10 mg/L of nanosilver meanwhile, hundred percent
inactivation occurred at 50 mg/L was dosed. While, Gong et al.,
(2007) successfully synthesized magnetic [email protected] nanoparticles. Such
particles size were ranging from 40 – 80 nm and they prove its ability to
inhibit E. coli (gram-negative), S. epidermidis (gram-positive)
growth. Therefore, this study aimed to evaluate a biocidal efficacy of calcium
hypochlorite as a common disinfectants used for water treatment and explore the
extent of its efficacy against waterborne pathogenic bacteria and trying to enhancement
its efficacy through using AgNPs after that a field trial was applied on the
basis of using a biocidal filter paper contains AgNPs loaded Ca(OCl)2 disinfectant
and evaluate its lethal effect on indicator bacterial counts for water


Materials and methods

Study area and period

This study was conducted on a private small cattle breeder farms located
in Beni-Suef (coordinates: 29°04’N31°05’E) and Fayoum
(coordinates: 29°308374?N–30°844105?E) province, Egypt throughout a period from May to December 2017.
Representative water samples were collected from both main source and water
troughs of both supplies (tap and hand pump water) at small cattle breeders (n=
15) in the study areas.

Study design

The protocol of this study includes two steps to investigate and improve
the hygienic quality of drinking water at small cattle breeders. The first one
aimed to evaluate the biocidal activity of calcium hypochlorite disinfectant
for drinking water and detect the extent of its efficacy against waterborne
bacteria then using nanocomposite contains silver nanoparticles (AgNPs) loaded bleaching
powder Ca(OCl)2 for trying to enhancement the disinfectant performance.
Stratified water samples were collected from small cattle breeder’s farms for
isolation and identification of pathogenic bacteria using biochemical tests.  The sensitivity of sixty bacterial strains was
evaluated with tested disinfectant, AgNPs, and AgNPs/Ca(OCl)2 composite
using broth macro dilution method. The second is a field trial for the sake of
evaluating the efficacy of bactericidal filter paper against indicators
coliforms bacteria. Water samples were bacteriologically examined prior and
post application of treated filter paper for total viable bacteria, total
coliform, and fecal coliform counts. All collected data were recorded and
statistically analyzed.

Water sampling

Under aseptic condition, a total of 100 water samples were collected
from both main source and water troughs used for cattle drinking at small
cattle breeders. The collected samples were obtained from two water supplies
(tap and hand pump water) in a 250 mL sterilized tightly capped bottle and
properly labeled. The outlet
of both water supplies was thoroughly disinfected using ethyl alcohol 70 %, and
then water samples were taken after allowed water flow. Samples were
transferred immediately to the lab in an ice box for further microbiological
examination according to standard water guidelines (APHA 2012).

Isolation and identification of bacterial pathogens

Samples were cultured on plate count agar (Lancashire BL97JJ, UK) for detection
and enumeration of total viable count (TVC), using pour plate method as described
by APHA (2012). Total coliform (TCC) was enumerated on M-Endo LES agar
(Difco, Sparks, MD) while fecal coliform (FCC) was counted on M-FC agar (EM
Science, Gibbstown, NJ) using membrane filtration (MF) technique according to APHA
(2012). Klebsiella spp. and E. coli were isolated on
MacConkey agar (Oxoid; CM 0115) and Eosine Methylene Blue (EMB) agar (Oxoid; CM
69) plates. While S. aureus was isolated on Baird Parker agar base (Oxoid
Ltd, Hampshire, England; CM 0275) with egg yolk supplement. Furthermore, for
purification, all isolated bacterial colonies are sub-cultured on nutrient agar
media. Enteric bacteria were identified on the basis of their colony,
morphology, and using API 20E (Biomerieux, Crappone France). S. aureus
strains were purified phenotypically by the tube coagulase test and
StaphID32API systems (API System, BioMe’rieux, Paris, France).

Evaluating the Biocidal activity of Calcium hypochlorite.

The selected disinfectant is a chemical substance (white powder,
contains 65% available chlorine and considered a strong oxidant) that used for disinfection
of drinking water due to being effective, easy to use, and stable. Furthermore,
it was proven effective under field conditions (Fuqua, 2010 and Lewis, 2010).
The efficacy of Ca(OCl)2 was tested against sixty
bacterial strains isolated from two different water supplies using broth macro
dilution method as described by Li et al. (2008) with minor changes
related to disinfectant concentrations and exposure times. The disinfectant was tested at different
concentrations (0.01 and 0.02 mg/L) and exposure times (30, 60, 120 and 180

Synthesis and characterization of AgNPs.

AgNPs were synthesized using chemical
reduction method according to Sileikaite et al. (2009). AgNPs were morphologically characterized by transmission electron
microscope (JEOL-JEM- 100CX II) in National Research Center (NRC), Egypt. The shape of Ag nanoparticles was spherical
and elongated. Furthermore, the diameter of nanoparticles (NP) is ranged
between 3.45 – 28.85 nm as shown in (Fig 1).

Evaluation methods of AgNPs and AgNPs/Ca(OCl)2 disinfectant

The bacterial isolates (sixty strains) were tested against AgNPs at a
concentration of 50 and 100 mg/L at different exposure times (10, 15, 30 and 60
mins) using broth macro dilution method. After that AgNPs/Ca(OCl)2 disinfectant
was prepared in order to an enhancement of its performance against tested
bacterial strains. 100 mg/L of AgNPs was added to 0.002 mg/L of Ca(OCl)2 disinfectant
at a ratio (1:2). The AgNPs/Ca(OCl)2 composite was shaking well
using magnetic stirrer for 4h continuously to avoid agglomeration of NP over
the incubation period then the nanocomposite centrifuged at 3000 rpm for 15
mins and washed by distilled water three times. Finally 100 ?l of freshly prepared isolated bacteria in trypticase broth (24h. growth) were subjected to 15
mL of AgNPs/ Ca(OCl)2 disinfectant at different concentrations (0.001 and 0.002 mg/L) and
exposure times (10, 15, 30
and 60 mins.) using broth macro dilution
method as described
by Li et al. (2008). Moreover, there were two conical flasks used as a control,
the first flask was contained inoculum and trypticase soya broth while the second
flask was contained AgNPs/ Ca(OCl)2  and trypticase soya
broth without inoculum.

Preparation of biocidal filter paper using AgNPs/Ca(OCl)2 disinfectant.

The filter paper is pure cellulose paper, porous and highly absorbent of
diameters (0.45mm, Sartorius Stedim Biotech GmbH, Germany). The porosity of
filter paper allows microorganisms to come into contact with AgNPs/Ca(OCl)2
disinfectant during water purification. The filter paper soaked overnight in 15
mL of AgNPs loaded Ca(OCl)2 disinfectant at a concentration of 0.002
mg/L then removed from the solution and rinsed by ethanol (70%) followed by soaking
in water for 5 minutes to remove the excess of unabsorbed nanocomposite and finally
drying the paper in an oven at 60° C for one hour. The shape of nanoparticles
and its distribution in filter paper was examined by TEM. Nanoparticles (NP)
diameter is ranged between 7.68 – 14.34 nm as shown in (Fig 2.)

Field trial for evaluating the biocidal filter paper.  

The biocidal activity of filter paper was tested against total viable (TVC)
and indicator coliforms bacteria (total and fecal coliform) counts in water
samples.  Twenty Representative water samples
were collected from water trough of both supplies. The samples were bacteriologically
examined prior and post-treatment throughout passing 100 mL of water samples on
both non-treated filter paper and AgNPs/Ca(OCl)2 filter paper for 10
minutes. All non-treated and treated filter paper were incubated on specific
agar media (M-Endo LES and M-FC agar) at 37°C for 24 hrs. After
that, the incubated plates were examined for identifying the absence and/or
growth of indicator bacteria (total and fecal coliform) counts in pre and post-treated
plates. Furthermore, all water samples were cultured for total viable counts (TVC)
on plate count agar. The targeted bacteria were enumerated on its specific media
as mention above to evaluate the efficacy and usability of biocidal filter
paper. Furthermore, effluent water (water drain after examined samples passing
on treated filter paper) was bacteriologically examined and the shape of
Coliforms bacteria in the effluent water was clarified using transmission
electron microscope.

Statistical analysis

All data were recorded using the Microsoft Excel spreadsheet then
prepared for analysis. The percentages of pathogenic bacteria isolated
from different water supplies in the investigated areas and the biocidal
activity of Ca(OCl)2, AgNPS and AgNPS /Ca(OCl)2 nanocomposite
against waterborne pathogenic bacteria were calculated using of
non-parametric tests (Chi-square test) using statistical package for social
sciences (SPSS, version 22.0).

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