This work evaluated the antimicrobial efficacy of kaolin clay and its loaded forms with carbon nanotubes (CNTs) and silver nanoparticles (AgNPs) against bacterial isolates from different water supplies (tap, underground and surface water) in addition to wastewater. A total of 160 water samples were collected from different water sources in the investigated districts. Samples were cultured for isolation and serological identification of pathogenic bacteria. AgNPs were synthesized by a typical one-step synthesis protocol, where CNTs were carried out in a reactor employing the double bias-assisted hot filament chemical vapor deposition method. Both were characterized using transmission electron microscopy, infrared and X-ray fluorescence (XRF) spectroscopy. The antimicrobial efficacy of each of natural kaolin clay, AgNPs- and CNTs-loaded clays were evaluated by their application in four concentrations (0.01, 0.03, 0.05 and 0.1 ppm) at different contact times (5 min, 15 min, 30 min and 2 h). AgNPs-loaded clays at concentrations of 0.05 and 0.1 mg/l for 2 h contact time exhibited a higher bactericidal efficacy on Escherichia coli and Salmonella spp. (70, 70, 80 and 90%, respectively) compared to CNTs-loaded clay. Concluding, the application of AgNPs-loaded clay for removal of water bacterial contaminants at a concentration of 0.1 ppm for 2 h contact times resulted in highly effective removals.

INTRODUCTION

There is no doubt that drinking water sources receive heavy loads of microorganisms through several means such as industrial, agricultural, and domestic wastes (Annual Drinking Water Quality Report 2005). A wide spectrum of pathogenic agents can be found in water and monitoring of their presence on a routine basis is impractical. Traditionally, microbial safety of drinking water has been confirmed by monitoring for the absence of microorganisms of fecal origin (Le-Chevallier & Au 2004).

To reduce the incidence of waterborne diseases and to make water suitable for drinking, removal of pathogenic organisms, fecal matters, suspended solids, algae, organic matters, and harmful chemicals from polluted water is mandatory (Gupta & Chaudhuri 1995). The removal capacity of a multiple-barrier water disinfection device for protozoa, bacteria, and viruses was studied and the efficiency of the multiple-barrier device in removing Escherichia coli was close to 100%, and more than 87% of Cryptosporidium oocysts and more than 98% of Giardia cysts were removed. Close to 100% of coliphages were removed and 99.6% of the adenovirus was removed (Espinosa-García et al. 2014). Moreover, for wastewater, stabilization ponds and constructed wetlands are efficient for the reduction of Cryptosporidium in wastewater, especially when the retention time is longer than 20 days with suitable sunlight and temperature while high rate filtration and chlorine disinfection are inefficient for the reduction of Cryptosporidium in effluents (Nasser 2016).

This study focused on nanotechnology and its applications which is considered as one of the rapidly developing sciences. As demand for fresh drinking water is increasing, nanotechnology can contribute notable development and improvement to water treatment processes. The disinfection process is the last and most important step in water and wastewater treatment processes. Some nanomaterials can be used as disinfectants due to their antimicrobial properties (Hossain et al. 2014). Carbon-based nanomaterials such as carbon nanotubes (CNTs), activated carbons, fullerene and graphene are widely used as the currently most promising functional materials due to their high adsorption capacities. Therefore, graphene nanosheets will challenge the current existing adsorbents, including other types of carbon-based nanomaterials (Yu et al. 2015).

Nano-sorbent materials such as CNTs and poly materials are applied to remove heavy metals, organics and biological impurities (Savage & Diallo 2005). CNTs have been gradually applied to the removal of organic contaminants (e.g., dyes, pesticides, and pharmaceuticals/drugs) from wastewater through adsorption processes (Yu et al. 2014), besides this, CNTs as adsorbent media have been proved to be able to remove a wide range of contaminants including bacteria (Akasaka & Watari 2009). CNTs with different diameters would have different specific surfaces, which would affect their adsorptive properties. However, the effects of different outer diameters of CNTs on adsorption of organic contaminates has not been investigated up to now (Yao et al. 2014).

Meanwhile, application of silver nanoparticles (AgNPs) has been extensively studied in the food industry for drinking water treatment (Konopka et al. 2009; Kumar & Raza 2009; Zhao et al. 2010). The antimicrobial activity of silver can be mainly attributed to interactions of silver ions with thiol groups of cellular proteins, leading to their inactivation. Processes such as cell respiration and ion transport across membranes, are also employed (Marambio-Jones & Van Hoek 2010). However, the mechanisms of toxic action for AgNPs are still not very well defined (Fabrega et al. 2011). The use of AgNPs in drinking water treatment has extensively become of interest (Feng et al. 2000; Jain & Pradeep 2005). However, addition of silver to water in such concentration that shows bactericidal activity does not impair the taste, color, odor, and other physicochemical characteristics of water (Klueh et al. 2000).

The present study aimed to evaluate the antimicrobial efficacy of natural kaolin clay and nano-adsorbent materials (CNTs and silver nitrate-loaded clay) against isolated bacteria from different water supplies (tap, underground and surface water) commonly used for human and animal drinking in addition to wastewater.

MATERIALS AND METHODS

Study area and period

A cross-sectional study was carried out in nine different rural areas in Beni-Suef Province (coordinates: 29°04'N 31°05'E), Egypt during the period from September 2014 to July 2015. These areas are suffering from health problems related to water quality on both human and animal levels.

Water sample collection

A total of 160 water samples were collected from main water sources intended for animal and human drinking. Samples represented three different water supplies (tap, underground [hand pumps], surface water [water channel] source) beside wastewater (n = 40 of each) in investigated districts. The tap and hand pump water samples were collected in 250 ml sterilized Schott Duran Bottles. For this purpose, the outlets of the taps and hand pumps were thoroughly disinfected by ethyl alcohol 70%, water was allowed to flow and then water samples were taken. Surface water and wastewater samples were collected 20 cm below the surface of the water using sterile plastic syringes (50 ml capacity) according to the method described previously (Azam et al. 2012). Sampling bottles were tightly capped, properly labeled and identified according to their source and site and immediately sent to the laboratory in an ice box for further microbiological examination according to standard guidelines (American Public Health Association (APHA) 2012).

Isolation and identification of bacterial pathogens

Isolation of bacterial pathogens

E. coli was isolated on MacConkey agar (Oxoid; CM 0115) and Eosin Methylene Blue agar (EMB; Oxoid; CM 69) plates meanwhile, Salmonella and Shigella species were isolated by pre-enrichment of water samples on buffer peptone water, then enrichment of samples on Selenite F broth, followed by isolation of the typical organism on selective medium, Xylose Lysine Deoxycholate agar (XLD) (Collee et al. 1996).

Biochemical and serological identification of bacterial pathogens

All colonies with different characteristics on EMB and XLD were sub-cultured onto nutrient agar for purification. Enteric bacteria isolated on respective selective or differential media were identified on the basis of their colony and morphological characteristics and then using API 20E (Biomerieux, Crappone France); final verification based on serological identification was carried out at the Ministry of Health, Cairo, Egypt, using techniques adopted by Sojka (1965). Serotyping was carried out by the slide agglutination technique using diagnostic polyvalent and monovalent E. coli O & K antisera.

Antimicrobial efficacy of natural kaolin and nano-adsorbent materials against isolated bacterial pathogens

The antimicrobial effects of natural kaolin clay and its loaded forms were evaluated against a total of 50 isolated bacterial pathogens from three different water supplies in addition to wastewater in the investigated districts.

Preparation of natural kaolin clay and its loaded forms

Natural clay pretreatment. Kaolin clay was collected from kilometre 18 of the Eastern Minia/Beni-Suef road. The pretreatment of kaolin was carried out by milling it to the sub-micro size. It was then washed with distilled water several times and after that dried in an oven at 100°C. Milling of the clay to the sub-micro size was then repeated, due to the agglomeration of clay particles which resulted from repeated washing and drying, and after that it was ready for use. Sub-micro sized clay agglomerated together to form micro granules after 10 h millings (1 μm in diameter) is shown in Figure 1.
Figure 1

Scanning electron microscope image of natural kaolin clay after 10 h milling.

Figure 1

Scanning electron microscope image of natural kaolin clay after 10 h milling.

CNTs-clay preparation. The composite of CNTs and clay was prepared by using kaolin as a catalyst during the synthesis of CNTs using the chemical vapor deposition technique (Pastorkova et al. 2012). The results indicated that CNTs clay composite was successfully fabricated and the clay and CNTs were not exfoliated by each other. The morphology of multi-walled carbon nanotubes (MWCNTs) kaolin clay composite was characterized by transmission electron microscope (TEM). Figure 2 shows the tubular structure of MWCNTs. Furthermore, it is clear that the outer walls of MWCNTs were deformed and amounts of amorphous carbon were deposited. From the high resolution TEM (HR-TEM) image of CNTs, the internal diameter of MWCNTs is approximately (7.06 nm) and the external diameter is nearly (15.66 nm).
Figure 2

TEM images for CNTs-clay composite at different magnifications.

Figure 2

TEM images for CNTs-clay composite at different magnifications.

Kaolin clay appears separated or linked with CNTs in Figure 3 which indicates that small amounts of clay are present inside CNTs due to their small nano-size (0.21 nm) and a large amount of clay is dispersed on the outer wall surface. These results revealed that the MWCNTs/kaolin composite are not exfoliated by each other (Pastorkova et al. 2012).
Figure 3

The X-ray diffraction pattern of CNTs-clay composite.

Figure 3

The X-ray diffraction pattern of CNTs-clay composite.

AgNPs-clay preparation. The effectiveness of AgNPs as an antimicrobial was determined. Whereas silver nitrate was used to investigate the effect of silver ions, stable AgNPs of <100 nm in size were synthesized through a one-step protocol according to the method described by Vigneshwaran et al. (2006) with some modifications. Five grams of natural clay were added to 100 ml of deionized water and heated to complete digestion, then 5 ml of 0.1 N solution of silver nitrate (AgNO3) (FW 169, 87 Gamma laboratory chemicals, Assay: Min 99.0%) were added and stirred well. This mixture was put into a dark glass bottle and autoclaved. After preparation of AgNPs-loaded clay, it was kept in a dark glass bottle away from direct sunlight at room temperature (25°C), and the size of AgNPs-loaded clay particles was measured by TEM (JEOL-JEM- 100CX II) and their size ranged from 5–40 μm (Figure 4). The elemental analysis by wavelength dispersive X-ray fluorescence (XRF) spectrometry revealed that the initial wt % of Ag2O (12.64%) has been added to the kaolin/AgNO3 loaded sample (Table 1), which was completely absent in the natural clay. This assures the successful loading of the clay with AgNO3. These data were confirmed through the observation of two peaks centered at energies very close to their tabulated line energies in the range 2.86–3.51 (Figure 5); one lies at 3.12 which is narrow and strong (1ry diffraction) with an intensity of 2.34 K and the other is less intense, lying at 3.25 with an intensity (2ry diffraction) of 1.04 K. Compared with the X-ray diffraction (XRD) pattern for the natural clay (Hassouna et al. 2014) such peaks are replaced by Si, Al, Ca and Mg ones that are normally found in clays. The total concentration of AgNPs stock was measured by graphite furnace atomic absorption (Model 210VGP) at the National Research Center, Cairo, Egypt.
Table 1

Elemental analysis of the kaolin/AgNO3 loaded sample by wavelength dispersive XRF spectrometry

Main constituents (wt %) Sample no. (1) 
SiO2 39.01 
TiO2 1.09 
Al2O3 19.21 
Fe2O3tot. 8.74 
MnO 0.02 
MgO 1.48 
CaO 2.76 
Na20.39 
K20.93 
Ag212.64 
P2O5 0.09 
SO3 0.04 
Cl 2.00 
L.O.I 11.39 
Cr2O3 0.028 
NiO 0.010 
CuO 0.008 
ZnO 0.026 
PbO 0.034 
Rb20.007 
Ga2O3 0.005 
As2O3 0.019 
Y2O3 0.010 
SrO 0.024 
Nb2O5 0.013 
ZrO2 0.034 
Main constituents (wt %) Sample no. (1) 
SiO2 39.01 
TiO2 1.09 
Al2O3 19.21 
Fe2O3tot. 8.74 
MnO 0.02 
MgO 1.48 
CaO 2.76 
Na20.39 
K20.93 
Ag212.64 
P2O5 0.09 
SO3 0.04 
Cl 2.00 
L.O.I 11.39 
Cr2O3 0.028 
NiO 0.010 
CuO 0.008 
ZnO 0.026 
PbO 0.034 
Rb20.007 
Ga2O3 0.005 
As2O3 0.019 
Y2O3 0.010 
SrO 0.024 
Nb2O5 0.013 
ZrO2 0.034 
Figure 4

TEM images for AgNPs-clay composite at different magnifications.

Figure 4

TEM images for AgNPs-clay composite at different magnifications.

Figure 5

The XRD pattern of AgNPs-clay composite.

Figure 5

The XRD pattern of AgNPs-clay composite.

Evaluation methods of natural kaolin and its loaded form

The antibacterial efficiency of kaolin clay and nano-adsorbent materials was determined using the broth macro dilution technique according to Li et al. (2008) with some modifications. Five sterile test tubes for each tested material (each test tube containing 1.2 ml homogeneous bacterial suspensions) were prepared in Brain Heart Infusion (BHI), then 0.8 ml of the tested material suspension were aseptically added to four test tubes to obtain final concentrations of 0.01, 0.03, 0.05 and 0.1 mg/l. Each of the natural kaolin, CNTs-loaded clay and AgNPs-loaded clay treatments were thoroughly mixed separately and allowed to interact with bacteria at different contact times (5, 15, 30 min and 2 h). The remaining test tube (the fifth one) was used as the negative control (1.2 ml bacterial suspension without any adsorbent material). Then, 1 ml of inocula was transferred to BHI-containing tubes, for incubation at 37 °C for 24 h. A sample was considered positive if it was exhibiting medium turbidity and the formation of a thin surface skin or of a precipitate in the bottom of the tube. After incubation, the suspension was spiked in solid medium (trypticase soya agar) to confirm the presence or absence of microorganisms tested against various tested materials and exposure times. The absence of bacterial growth in plates indicates the effectiveness of the adsorbent.

Statistical analysis

The data collected and recorded on specifically designed forms were entered in the Microsoft Excel spreadsheet then prepared for analysis. The frequent distribution of bacterial isolates from different water sources was calculated by using percentage values and analyzed using a Chi-square test (SPSS, version, 22).

RESULTS

The frequent distribution of pathogenic bacteria isolated from different water sources in (Table 2) clarified that there were in total 121 bacterial isolates out of 160 examined samples. The highest percentages of bacterial contaminants were isolated from waste and surface water sources (100% of each) at X2 = 9.52 (P < 0.05), while the lowest percentages were presented in underground and tap water (20% and 5% respectively). Moreover, the predominant bacterial isolates were E. coli and Shigella flexneri followed by Salmonella spp., Klebsiella aerogenes and Klebsiella pneumoniae (34.7, 24.7, 16.5, 14.0 and 9.9%, respectively).

Table 2

The frequency distribution of pathogenic bacteria isolated from different water sources

Bacteriological finding Total Bacterial isolates (%) 
      Klebsiella Shigella 
Water samples Examined (No.) Positive No. (%) Total (No.) E. coli Salmonella spp. pneumoniae aerogenes flexneri 
Tap 40 5.0 ND ND 20 60 20 
Underground 40 20.0 12 16.6 16.6 ND 33.3 33.3 
Surface 40 100 46 28.2 15.2 17.3 10.8 28.2 
Waste 40 100 58 46.5 18.9 5.1 8.6 20.6 
Total 160  121 34.7 16.5 9.9 14.0 24.7 
Bacteriological finding Total Bacterial isolates (%) 
      Klebsiella Shigella 
Water samples Examined (No.) Positive No. (%) Total (No.) E. coli Salmonella spp. pneumoniae aerogenes flexneri 
Tap 40 5.0 ND ND 20 60 20 
Underground 40 20.0 12 16.6 16.6 ND 33.3 33.3 
Surface 40 100 46 28.2 15.2 17.3 10.8 28.2 
Waste 40 100 58 46.5 18.9 5.1 8.6 20.6 
Total 160  121 34.7 16.5 9.9 14.0 24.7 

ND: not detected, X2 = 9.52, P < 0.05.

Concerning serological identification of E. coli isolated from different water sources (Table 3), it has been found that the predominant E. coli serotypes in different water sources were O128:k67, O157:k-, O111:k58 and O55:k59. The highest serotype percentages in wastewater were O157:k- and O111:k58 (33.3% and 25.9%, respectively) followed by O128:k67 and O55:k59 (18.55% of each). However, in surface water, the highest percentage of E. coli serotype was O157:k- followed by O128:k67, O55:k59 and O111:k58 (30.7, 23.07, 23.07 and 15.3%, respectively) while in underground water, O128:k67 and O111:k58 accounted for 50% each.

Table 3

Serological identification of E. coli isolated from different water sources

Isolated bacteria  E. coli serotypes (%) 
Water samples Total isolated (No.) O128:k67 O157:k- O111:k58 O55:k59 
Tap ND ND ND ND 
Underground 1 (50) ND 1 (50) ND 
Surface 13 3 (23.07) 4 (30.7) 2 (15.3) 3 (23.07) 
Waste 27 5 (18.5) 9 (33.3) 7 (25.9) 5 (18.5) 
Total 42 11 (26.19) 13(30.9) 10 (23.8) 8 (19.04) 
Isolated bacteria  E. coli serotypes (%) 
Water samples Total isolated (No.) O128:k67 O157:k- O111:k58 O55:k59 
Tap ND ND ND ND 
Underground 1 (50) ND 1 (50) ND 
Surface 13 3 (23.07) 4 (30.7) 2 (15.3) 3 (23.07) 
Waste 27 5 (18.5) 9 (33.3) 7 (25.9) 5 (18.5) 
Total 42 11 (26.19) 13(30.9) 10 (23.8) 8 (19.04) 

ND: not detected.

A comparison of the antimicrobial efficacy of natural kaolin clay with nano-adsorbent materials against bacterial isolates from different water sources is presented in Table 4. It has been revealed that AgNPs-loaded clay at a concentration of 0.1 mg/l had the highest antimicrobial activity against Salmonella spp. (90%) followed by E. coli, Klebsiella pneumoniae and Shigella flexneri (80% each) and Klebsiella aerogenes (70%) after 2 h of exposure time. The antimicrobial activity of CNTs-loaded clay at the same concentration was 70% for each of Salmonella spp. and Klebsiella pneumoniae, while it was 60% for E. coli isolates.

Table 4

Antimicrobial efficacy of natural clay and nano-adsorbent compounds against isolated bacteria of different water sources after two-hour exposure time

Bacterial isolates (spp.) E. coli Salmonella spp. Klebsiella Shigella flexneri 
Tested compounds       pneumoniae aerogenes    
dose(ppm) 
Natural clay (2.0) 0.0 0.0 100 0.0 0.0 100 0.0 0.0 100 0.0 0.0 100 0.0 0.0 100 
CNTs-clay (0.1) 60 10 30 70 10 20 70 20 10 60 10 30 40 40 20 
AgNPs-clay (0.1) 80 10 10 90 10 0.0 80 20 0.0 70 20 10 80 10 10 
Bacterial isolates (spp.) E. coli Salmonella spp. Klebsiella Shigella flexneri 
Tested compounds       pneumoniae aerogenes    
dose(ppm) 
Natural clay (2.0) 0.0 0.0 100 0.0 0.0 100 0.0 0.0 100 0.0 0.0 100 0.0 0.0 100 
CNTs-clay (0.1) 60 10 30 70 10 20 70 20 10 60 10 30 40 40 20 
AgNPs-clay (0.1) 80 10 10 90 10 0.0 80 20 0.0 70 20 10 80 10 10 

S: Sensitive (absence of bacterial growth), I: Intermediate (moderate bacterial growth), R: Resistant (bacterial growth).

The results in Table 5 show the antimicrobial efficacy of AgNPs-loaded clay at different exposure times. It was found that the highest antimicrobial activity was achieved at the concentration dose of 0.1 mg/l and 2 h exposure time for all tested bacterial isolates (Salmonella spp. was 90%, followed by E. coli, Klebsiella pneumoniae and Shigella flexneri at 80% each). Meanwhile all bacterial isolates showed a highly resistant profile to other concentrations (0.01, 0.03 and 0.05 mg/l) at different exposure times (5 min, 15 min, and 30 min, respectively). The presented data in Table 6 clarify that the antimicrobial efficacy of CNTs-clay was higher against Salmonella spp. and Klebsiella pneumoniae (70% for each) compared to E. coli, Klebsiella aerogenes and Shigella flexneri (60, 60 and 40%, respectively). Moreover, the same bacterial isolates revealed that highly resistant patterns were present at low tested concentrations of CNTs at different exposure times.

Table 5

Antimicrobial efficacy of AgNPs-loaded clay against isolated bacteria from different water sources at different exposure times

  Antimicrobial activity (%) of AgNPs-clay at different exposure times 
  5 min 15 min 30 min 2 h 
Bacterial isolates (spp.) Doses (mg/l) 
E. coli 0.01 0.0 0.0 100 0.0 0.0 100 0.0 0.0 100 0.0 20 80 
0.03 0.0 0.0 100 0.0 0.0 100 0.0 40 60 0.0 40 60 
0.05 0.0 0.0 100 0.0 20 80 20 50 30 70 10 20 
0.1 0.0 20 80 0.0 40 60 80 20 20 80 10 10 
Salmonella spp. 0.01 0.0 0.0 100 30 10 60 30 20.0 50 40 30 30 
0.03 0.0 0.0 100 40 20 40 30 40 30 60 30 10 
0.05 0.0 0.0 100 60 20 20 60 30 10 70 20 10 
0.1 0.0 20 80 70 0.0 30 70 20 10 90 10 0.0 
Shigella flexneri 0.01 0.0 0.0 100 0.0 0.0 100 0.0 0.0 100 0.0 30 70 
0.03 0.0 0.0 100 0.0 0.0 100 0.0 50 50 0.0 50 50 
0.05 0.0 0.0 100 0.0 40 60 50 40 10 70 20 10 
0.1 0.0 20 80 0.0 50 50 70 20 10 80 10 10 
K. pneumoniae 0.01 0.0 0.0 100 0.0 0.0 100 0.0 30 70 0.0 40 60 
0.03 0.0 0.0 100 0.0 0.0 100 0.0 40 60 0.0 50 50 
0.05 0.0 0.0 100 0.0 60 40 60 20 20 60 30 10 
0.1 0.0 40 60 0.0 50 50 60 30 10 80 20 0.0 
K. aerogenes 0.01 0.0 0.0 100 0.0 0.0 100 0.0 20 80 0.0 20 80 
0.03 0.0 0.0 100 0.0 0.0 100 0.0 40 60 0.0 40 60 
0.05 0.0 0.0 100 0.0 50 50 40 20 40 50 20 30 
0.1 0.0 30 70 0.0 40 60 30 50 20 70 20 10 
  Antimicrobial activity (%) of AgNPs-clay at different exposure times 
  5 min 15 min 30 min 2 h 
Bacterial isolates (spp.) Doses (mg/l) 
E. coli 0.01 0.0 0.0 100 0.0 0.0 100 0.0 0.0 100 0.0 20 80 
0.03 0.0 0.0 100 0.0 0.0 100 0.0 40 60 0.0 40 60 
0.05 0.0 0.0 100 0.0 20 80 20 50 30 70 10 20 
0.1 0.0 20 80 0.0 40 60 80 20 20 80 10 10 
Salmonella spp. 0.01 0.0 0.0 100 30 10 60 30 20.0 50 40 30 30 
0.03 0.0 0.0 100 40 20 40 30 40 30 60 30 10 
0.05 0.0 0.0 100 60 20 20 60 30 10 70 20 10 
0.1 0.0 20 80 70 0.0 30 70 20 10 90 10 0.0 
Shigella flexneri 0.01 0.0 0.0 100 0.0 0.0 100 0.0 0.0 100 0.0 30 70 
0.03 0.0 0.0 100 0.0 0.0 100 0.0 50 50 0.0 50 50 
0.05 0.0 0.0 100 0.0 40 60 50 40 10 70 20 10 
0.1 0.0 20 80 0.0 50 50 70 20 10 80 10 10 
K. pneumoniae 0.01 0.0 0.0 100 0.0 0.0 100 0.0 30 70 0.0 40 60 
0.03 0.0 0.0 100 0.0 0.0 100 0.0 40 60 0.0 50 50 
0.05 0.0 0.0 100 0.0 60 40 60 20 20 60 30 10 
0.1 0.0 40 60 0.0 50 50 60 30 10 80 20 0.0 
K. aerogenes 0.01 0.0 0.0 100 0.0 0.0 100 0.0 20 80 0.0 20 80 
0.03 0.0 0.0 100 0.0 0.0 100 0.0 40 60 0.0 40 60 
0.05 0.0 0.0 100 0.0 50 50 40 20 40 50 20 30 
0.1 0.0 30 70 0.0 40 60 30 50 20 70 20 10 

S: Sensitive (absence of bacterial growth), I: Intermediate (moderate bacterial growth), R: Resistant (bacterial growth).

Table 6

Antimicrobial efficacy of CNTS-loaded clay against isolated bacteria from different water sources at different exposure times

  Antimicrobial activity (%) of CNTS-clay at different exposure times 
  5 min 15 min 30 min 2 h 
Bacterial isolates (spp.) Doses (mg/l) 
E. coli 0.01 0.0 0.0 100 0.0 0.0 100 0.0 0.0 100 0.0 0.0 100 
0.03 0.0 0.0 100 0.0 0.0 100 0.0 0.0 100 0.0 0.0 100 
0.05 0.0 0.0 100 0.0 0.0 100 0.0 0.0 100 0.0 0.0 100 
0.1 0.0 0.0 100 0.0 0.0 100 0.0 0.0 100 60 10 30 
Salmonella spp. 0.01 0.0 0.0 100 0.0 0.0 100 0.0 0.0 100 0.0 0.0 100 
0.03 0.0 0.0 100 0.0 0.0 100 0.0 0.0 100 0.0 0.0 100 
0.05 0.0 0.0 100 0.0 0.0 100 0.0 0.0 100 0.0 0.0 100 
0.1 0.0 0.0 100 0.0 0.0 100 0.0 0.0 100 70 10 20 
Shigella flexneri 0.01 0.0 0.0 100 0.0 0.0 100 0.0 0.0 100 0.0 0.0 100 
0.03 0.0 0.0 100 0.0 0.0 100 0.0 0.0 100 0.0 0.0 100 
0.05 0.0 0.0 100 0.0 0.0 100 0.0 0.0 100 30 10 50 
0.1 0.0 0.0 100 0.0 0.0 100 0.0 0.0 100 40 40 20 
K. pneumoniae 0.01 0.0 0.0 100 0.0 0.0 100 0.0 0.0 100 0.0 0.0 100 
0.03 0.0 0.0 100 0.0 0.0 100 0.0 0.0 100 0.0 0.0 100 
0.05 0.0 0.0 100 0.0 0.0 100 0.0 0.0 100 40 50 10 
0.1 0.0 0.0 100 0.0 0.0 100 0.0 0.0 100 70 20 10 
K. aerogenes 0.01 0.0 0.0 100 0.0 0.0 100 0.0 0.0 100 0.0 0.0 100 
0.03 0.0 0.0 100 0.0 0.0 100 0.0 0.0 100 0.0 0.0 100 
0.05 0.0 0.00 100 0.0 0.0 100 0.0 0.0 100 40 20 40 
0.1 0.0 0.0 100 0.0 0.0 100 0.0 0.0 100 60 10 30 
  Antimicrobial activity (%) of CNTS-clay at different exposure times 
  5 min 15 min 30 min 2 h 
Bacterial isolates (spp.) Doses (mg/l) 
E. coli 0.01 0.0 0.0 100 0.0 0.0 100 0.0 0.0 100 0.0 0.0 100 
0.03 0.0 0.0 100 0.0 0.0 100 0.0 0.0 100 0.0 0.0 100 
0.05 0.0 0.0 100 0.0 0.0 100 0.0 0.0 100 0.0 0.0 100 
0.1 0.0 0.0 100 0.0 0.0 100 0.0 0.0 100 60 10 30 
Salmonella spp. 0.01 0.0 0.0 100 0.0 0.0 100 0.0 0.0 100 0.0 0.0 100 
0.03 0.0 0.0 100 0.0 0.0 100 0.0 0.0 100 0.0 0.0 100 
0.05 0.0 0.0 100 0.0 0.0 100 0.0 0.0 100 0.0 0.0 100 
0.1 0.0 0.0 100 0.0 0.0 100 0.0 0.0 100 70 10 20 
Shigella flexneri 0.01 0.0 0.0 100 0.0 0.0 100 0.0 0.0 100 0.0 0.0 100 
0.03 0.0 0.0 100 0.0 0.0 100 0.0 0.0 100 0.0 0.0 100 
0.05 0.0 0.0 100 0.0 0.0 100 0.0 0.0 100 30 10 50 
0.1 0.0 0.0 100 0.0 0.0 100 0.0 0.0 100 40 40 20 
K. pneumoniae 0.01 0.0 0.0 100 0.0 0.0 100 0.0 0.0 100 0.0 0.0 100 
0.03 0.0 0.0 100 0.0 0.0 100 0.0 0.0 100 0.0 0.0 100 
0.05 0.0 0.0 100 0.0 0.0 100 0.0 0.0 100 40 50 10 
0.1 0.0 0.0 100 0.0 0.0 100 0.0 0.0 100 70 20 10 
K. aerogenes 0.01 0.0 0.0 100 0.0 0.0 100 0.0 0.0 100 0.0 0.0 100 
0.03 0.0 0.0 100 0.0 0.0 100 0.0 0.0 100 0.0 0.0 100 
0.05 0.0 0.00 100 0.0 0.0 100 0.0 0.0 100 40 20 40 
0.1 0.0 0.0 100 0.0 0.0 100 0.0 0.0 100 60 10 30 

S: Sensitive (absence of bacterial growth), I: Intermediate (moderate bacterial growth), R: Resistant (bacterial growth).

DISCUSSION

Drinking water has the potentiality to transport microbial pathogens to a great number of individuals, causing subsequent illness, which is well documented in countries at all levels of economic development (Payment 1997). In this study, results revealed that the highest percentages of bacterial isolates (E. coli and Salmonella spp.) were observed in wastewater followed by surface water supply as compared to other water sources in the investigated districts. These results could be attributed to the impact of unsupervised anthropogenic activities including excessive usage of fertilizers and pesticide, waste disposal and industrial waste, seepage from septic tanks and construction of water pipes. The surface water presented significant acute toxicity, and could cause diseases if used in livestock farms without being adequately treated. This represents a dangerous public health risk, which needs future evaluation and control. This was in agreement with Melegy et al. (2014) who showed that pathogenic bacteria can occur in surface water in large numbers, either being excreted in feces or occurring naturally in the environment. Moreover, Mohammed (2016) revealed that in surface water, E. coli was isolated in the highest percentage (17/56; 56.7%) followed by Staph. aureus (11/56; 36.7%), Salmonella spp. (8/56; 26.7%), Strept. faecalis (7/39; 23.3%), Shigella flexneri (5/56; 16.7%), Proteus spp. (5/56; 16.7%) and Klebsiella pneumoniae (3/56; 10.0%) at X2 = 9, P ≤ 0.01. Sources of fecal contamination include humans, livestock and wild animals; in general, human fecal waste gives rise to the highest risk of waterborne disease (Craun 1996). The most sporadic cases of waterborne intestinal illness will not be detected, or if detected may not be recognized as water-related, while it is estimated that water, sanitation, and hygiene are responsible for 40% of all deaths and 5.7% of the total disease burden occurring worldwide (Prȕss et al. 2002).

E. coli is a widely used bio-indicator of fecal contamination in water sources. External contact and subsequent ingestion of bacteria from fecal contamination can cause detrimental health effects (Money et al. 2009). In this study, it has been found that the predominant E. coli serotypes in different water sources were O128:k67, O157:k-, O111:k58 and O55:k59. Environmental Protection Agency and epidemiological investigations, suggested that cultural fecal indicator counts are valid predictors of disease risk (Sinton et al. 1994) and sewage can serve as a vehicle for bacteria to enter into human and non-human hosts either by direct contact or through contamination of drinking water supplies (Boczek et al. 2007).

The current increased attention is directed to health problems associated with drinking water quality at both human and animal level. In particular, communities who use surface water supplies are of special concern, since their drinking water is commonly not treated or monitored for contamination on a routine basis. The present study evaluated the efficiency of natural kaolin and nano-adsorbent materials for the removal of water contaminants, and subsequently risk hazards to human and animal health. The present study revealed that different types of microorganisms vary in their response to the tested material of kaolin clay, CNTs-loaded clay, and AgNPs-loaded clay. Natural kaolin clay was the least effective against all bacterial pathogens. The results also suggested that the antimicrobial effects of kaolin and nano-adsorbent materials are not only dependent on the types of material but also on their concentrations and exposure times. A previous study showed that AgNPs supported on granular activated carbon gave good results against Escherichia coli (Acevedo et al. 2014), instead, in the present work we used kaolin as a support material which is highly available and cost effective. The efficiency of AgNPs-clay was highly effective against bacterial pathogens at concentrations of 0.05 and 0.1 mg/l. These results are in agreement with Sondi & Salopek-Sondi (2004) who suggested that the antimicrobial effects of the AgNPs on bacteria were dependent on the concentration of Ag in the nanoparticles (NPs); where increasing the concentration of NPs delayed the growth of bacteria. The treated bacterial cells were significantly changed and showed major damage, which was characterized by the formation of ‘pits’ in their cell walls. Previous studies have shown that antimicrobial formulations in the form of NPs could be used as effective bactericidal materials due to their enhanced reactivity, resulting from their high surface/volume ratio (Pal et al. 2007; Choi et al. 2008; Park et al. 2009). Particularly, silver in the form of NP (AgNPs) is known to exhibit strong biocidal effects on different bacterial species (Sondi & Salopek-Sondi 2004; Rai et al. 2009). Moreover, silver ions and silver-based compounds are highly toxic to Gram-negative and Gram-positive microorganisms (Bae et al. 2010; Fayaz et al. 2010; You et al. 2011; Maillard & Hartemann 2013).

CNTs are graphene sheets rolled into a tube and possibly capped by half a fullerene. They can be either single-walled (SWNTs), a single pipe with a diameter from 1 to 5 nm, or multi-walled (MWNTs), with several nested tubes, at lengths varying from 100 nm up to several tens of micrometers (Li et al. 2008). In the present study, the antimicrobial activity of CNTs against bacterial isolates from different water sources revealed that the sensitivities (%) of almost all bacterial pathogens to CNTs were 70% after 2 h exposure time.

However, the bacterial isolates which showed resistant to CNTs were E. coli, Klebsiella aerogenes (30% each), Salmonella spp., Shigella flexneri (20% each) and Klebsiella pneumoniae (10%) after 2 h exposure time. The results could be interpreted to suggest that the main factors which controlled their sensitivity were nanomaterial (CNTs) concentration and contact times. These results are supported by Kang et al. (2007) who clarified that antimicrobial activity of CNTs requires direct contact between CNTs and target microorganisms. Suspension of non-functionalized CNTs in water is extremely difficult and does not provide enough CNT-microbe contact for disinfection. Accordingly, the antibacterial activity of CNTs could be exploited by coating CNTs on a reactor surface in contact with the pathogen-laden water, also it has been found that using immobilized SWNTs on a membrane filter surface led to 87% killing of E. coli in a 2 h contact time.

CONCLUSION

Based on the present results, it can be concluded that the application of AgNPs-loaded clay for removal of water bacterial contaminants resulted in highly effective removals at a concentration of 0.1 ppm and 2 h contact time, and that CNTs-loaded clay was also effective, but to a lesser extent, at the same concentration and exposure time.

ACKNOWLEDGEMENTS

The authors would like to acknowledge the Projects Funding and Granting Unit in Beni-Suef University, Egypt for funding the project and supporting the research.

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