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International Journal of ChemTech Research CODEN (USA): IJCRGG, ISSN: 0974-4290, ISSN(Online):2455-9555 Vol.9, No.11 pp 287-298, 2016
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Synthesis and applications of silver nanoparticles on bacterial pathogens activity
B. A. Almayahi*, Laith B. Alhusseini
Department of Environment, College of Science, University of Kufa, Najaf, Iraq
Abstract : This research focused on the study effect silver nanoparticles (AgNPs) on bacterial activity. Silver nanoparticle concentrationsare (8, 6) mM. The results showed that the best method to prepare the silver nanoparticles was sunlight method. It concluded that concentration of 8 mM better than 6 mM for processing bacterial activity.The silvernanoparticles are succeeded to inhibit the growth of pathogenic bacteria examined in this study.
Keyword : AgNPs, antibacterial, bacterial pathogens.
Introduction
Nanoparticles are attracted much attention because of their unique size-dependent optical, magnetic, and catalytic properties1. Metal nanoparticles have intensively studied within the past years2. Nano materials have an important subject in basic and applied sciences for their applications in wide ranges of different fields, including chemistry, physics, biology, materials science, medicine, and catalysis3. Metal nanoparticles are prepared using different methods such as laser ablation technique, chemical reduction or silver salt, photo-reduction, microorganisms, arc-Discharge and bio surfactant3-9.The development of new resistant strains of bacteria to current antibiotics has become a serious problem in public health; there is a strong incentive to develop new bactericides. Silver has long known to exhibit a strong toxicity to a wide range of microorganisms for this reason silver-based compound is used in many bactericidal applications10. Several salts of silver and their derivatives are employed as antimicrobial agents11. The bactericidal property of these nanoparticles depends on it stability in the growth medium, this imparts greater retention time for bacterium–nanoparticle interaction. There lies a strong challenge in preparing nanoparticles of silver stable enough significantly restrict bacterial growth. The use of nanoparticles of metals is a viable solution to stop infectious diseases due to the antimicrobial properties of nanoparticles. The growth inhibition relate to the formation of free radicals from the surface of Ag. Uncontrolled generation of free radicals can attack membrane lipids and then lead to a breakdown of membrane function. The major mechanism through which AgNps manifested antibacterial properties is by anchoring to and penetrating the bacterial cell walls, and modulating cellular signaling12. AgNpsact primarily in three ways against bacteria:
Silver metal is the effective in preventing bacterial infection of wounds. Pathogens bacteria types namely13, 14: Escherichia coli(E. coli) is a gram-negative, facultative anaerobic, rod-shaped bacterium of the
genus Escherichia that found in the lower intestine of warm-blooded organisms. Most E. coli strains are harmless, but some serotypes can cause serious food poisoning in their hosts, Klebsiella is a genus of no motile of gram-negative , oxidase-negative , and rod-shaped bacteria with a prominent polysaccharide -based capsule 15, 16.Bacillus is a genus of gram-positive, rod-shaped (bacillus) bacteria, and a member of the phylum Firmicutes. Bacillus species can be obligate aerobes (oxygen reliant), or facultative anaerobes. Pseudomonas is a genus of gram-negative and aerobic gamma proteobacteria, belonging to the family Pseudomonadaceae containing 191 validly described species. Proteus is a genus of gram-negative Proteobacteria . Proteus bacilli are widely distributed in nature as saprophytes, found in decomposing animal matter, sewage, manure soil, and human and animal feces. They are opportunistic pathogens, commonly responsible for urinary and septic infections, often nosocomial . Serratia is a genus of gram-negative, facultative anaerobic and rod-shaped bacteria of the Enterobacteriaceae family. The most common species in the genus, S.marcescens, is the only pathogen and usually causes nosocomial infections. S. odoriferae have caused diseases through infection; Staphylococcus is a genus of gram-positive bacteria . Under the microscope , they appear round, and form in grape -like clusters. The Staphylococcus genus includes at least 40 species. Nine have two subspecies , one has three subspecies and one has four subspecies. Most are harmless and reside normally on the skin and mucous membranes of humans and other organisms . They are a small component of soil microbial flora . These bacteria are found everywhere in nature. They can be found in water, soil, plants, insects, animals, and humans17-20. Ubiquitous in nature, Bacillus includes both free-living and parasitic pathogenic species and under stressful environmental conditions. The present work demonstrates the effect of AgNPs on the bacterial activity against seven Gram-positive and Gram-negative isolates.
Materials and Methods
Seven bacteria samples (E.coli, klebsiella, bacillus, pseudomonas, Proteus, Serratia, staphylococcus) collected from Alforat and Alsadr hospitals in Najf city, Iraq as shown in Fig. 1a. These bacteria are used with serial dilution in the pipe to identify the AgNps efficiency of bacterial growth inhibitory (antibacterial). This method is showed the effect of AgNpson grams negative and positive bacteria. 28 g nutrient ager dissolved in 1 L distilled water mixed in two conical flasks each one 500 ml and closed by cotton, then flasks rotate about benzene lamp to get a clear color to 30 m, after that insert the flasks in autoclave (15 min, 15 bar, 121oC) as shown in Fig. 1b. These bacteria has cooled and cultivated in 42 Petri dishes. Six dishes with nutrient agar (after cooling) are used for seven bacteria types, every type of 1 ml moved using syringe to dish by dilution (10-3 of 3 dish's) and (10-4of 3 dish's)as shown in Fig. 1c.Aqueous solution of silver nitrate (AgNO3) at concentration of (8, 6) mM was prepared and used for the synthesis of silver nanoparticles after exposed it to bright sunlight at 50 °C; the change of solution color within few minutes. Atomic absorption spectroscopy iscarried out for the estimation of silver concentration in the prepared silver nanoparticles solution.Then (3-4) drops from AgNps added to 4 dish's using syringe to each type. Two dishes of dilution (10-3, 10-4) treated by AgNps of 8 mM, two dishes of dilution (10-3, 10-4) treated by AgNps of 6 Mm, andtwodishes without treatment are inserted in incubation as shown in Fig. 1d.The dishes are incubated at 37°C for 24 h at 37 oC.Bacteria colonies are measured using direct counting colonies.
Fig. 1 Procedure for preparation and treatment using AgNps
Results
Table 1 shows the number of bacterial colonies obtained from treating bacterial using AgNps. Table 1alsoshows colonies number of each bacterium at dilution 10-3 and 10-4 without treatment. Whereas, no detected any colonies of bacteria at dilution 10-3 and 10-4, when it treatment with AgNps at concentrations (8 and 6mM). When bacteria are treatment with AgNps of (6) mM found 7 Proteus colonies at dilution of 10-3this means that the concentration (8) mM better than 6 mM in process for bacterial activity. Highest number of colonies (286 colonies) is obtained in pseudomonas type at dilution (10-3) without AgNPs. Whereas, the lowest number (7 colonies)is obtained of Proteus at dilution (10-3) and concentration (6mM).
Table 1Bacteria types with dilution
Bacteria type |
Dilution (10-4) 6mM |
Dilution (10-4) 8mM |
Dilution (10-3) 6mM |
Dilution (10-3) 8Mm |
Control Dilution (10-4) |
Control Dilution (10-3) |
E.coli |
ND |
ND |
ND |
ND |
07 |
401 |
Klebsiella |
ND |
ND |
ND |
ND |
400 |
202 |
Bacillus |
ND |
ND |
ND |
ND |
411 |
284 |
Pseudomonas |
ND |
ND |
ND |
ND |
467 |
286 |
Proteus |
ND |
ND |
7 |
ND |
88 |
411 |
Serratia |
ND |
ND |
ND |
ND |
418 |
271 |
ND: No detected.
Fig. 2 shows pseudomonas bacterial before and after treatment using AgNps concentrations (6 and 8) mM. The colonies are grown without treatment at dilutions 10-3and 10-4as shown in Figs. 2A, B. The colonies are not detected ,when treated with AgNPs concentration(6) mM at dilutions 10-3and 10-4as shown in Figs. 2A1, B1. The colonies are not detected with AgNPs concentration (8) mM as shown in Figs. 2A2, B2.
Fig. 2: pseudomonas bacteria: (A) Dilution 10-3 without AgNPs. (A1) Dilution 10-3 with AgNPs of 6mM.(A2) Dilution 10-3 with AgNPs of 8mM. (B) Dilution 10-4 without AgNPs. (B1) Dilution 10-4 with AgNPs of 6mM. (B2) Dilution 10-4with AgNPs of 8mM.
Fig. 3 shows Proteus bacterial before and after treatment by AgNpsof different concentrations (6 and 8) mM. The colonies are grown without treatment at dilutions 10-3 and 10-4 as shown in Figs. 3A, B. whereas, the low number (7 colonies) is obtained of Proteus at dilution (10-3) with concentration (6 mM) as shown in Fig. 3 A1. The colonies are not detected, when treated with AgNPs concentration (6) mM at dilutions10-4 as shown in Fig. 3B1.The colonies are not detected with AgNPs concentration (8) mM as shown in Figs. 3A2, B2.
Fig. 3: Proteus bacteria: (A) Dilution 10-3 without AgNPs. (A1) Dilution 10-3 with AgNPs of 6mM.(A2) Dilution 10-3 with AgNPs of 8mM. (B) Dilution 10-4 without AgNPs. (B1) Dilution 10-4 with AgNPs of 6mM. (B2) Dilution 10-4with AgNPs of 8mM.
Fig. 4 shows Klebsiella bacterial before and after treatment by AgNps in different concentrations (6 and 8) mM. The colonies are grown without treatment at dilutions 10-3and 10-4as shown in Figs. 4A, B. The colonies are not detected, when treated with AgNPs concentration (6) mMat dilutions 10-3and 10-4as shown in Figs. 4A1, B1. The colonies are not detected with AgNPs concentration (8) mM as shown in Figs. 4A2, B2.
Fig. 4: Klebsiella bacteria: (A) Dilution 10-3 without AgNPs. (A1) Dilution 10-3 with AgNPs of 6mM.(A2) Dilution 10-3 with AgNPs of 8mM. (B) Dilution 10-4 without AgNPs. (B1) Dilution 10-4 with AgNPs of 6mM. (B2) Dilution 10-4with AgNPs of 8mM
Fig. 5 shows E.coli bacterial before and after treatment by AgNpsin different concentrations (6 and 8) mM. The colonies are grown without treatment at dilutions 10-3and 10-4as shown in Figs. 5A, B. The colonies are not detected, when treated with AgNPs concentration (6) mMat dilutions 10-3and 10-4as shown in Figs. 5A1, B1. The colonies are not detected with AgNPs concentration (8) mM as shown in Figs. 5A2, B2.
Fig. 5: E.coli bacteria: (A) Dilution 10-3 without AgNPs. (A1) Dilution 10-3 with AgNPs of 6mM.(A2) Dilution 10-3 with AgNPs of 8mM. (B) Dilution 10-4 without AgNPs. (B1) Dilution 10-4 with AgNPs of 6mM. (B2) Dilution 10-4with AgNPs of 8mM
Fig. 6 shows Bacillu sbacterial before and after treatment by AgNps in different concentrations (6 and 8) mM. The colonies are grown without treatment at dilutions 10-3and 10-4as shown in Figs. 6A, B. The colonies are not detected, when treated with AgNPs concentration (6) mMat dilutions 10-3and 10-4as shown in Figs. 6A1, B1. The colonies are not detected withAgNPs concentration (8) mM as shown in Figs. 6A2, B2.
Fig. 6: Bacillus bacteria: (A) Dilution 10-3 without AgNPs. (A1) Dilution 10-3 with AgNPs of 6mM.(A2) Dilution 10-3 with AgNPs of 8mM. (B) Dilution 10-4 without AgNPs. (B1) Dilution 10-4 with AgNPs of 6mM. (B2) Dilution 10-4with AgNPs of 8mM
Fig. 7 shows Serratia bacterial before and after treatment by AgNps in different concentrations (6 and 8) mM. The colonies are grown without treatment at dilutions 10-3and 10-4as shown in Figs. 7A, B. The colonies are not detected, when treated with AgNPs concentration (6) mMat dilutions 10-3and 10-4as shown in Figs. 7A1, B1. The colonies are not detected with AgNPs concentration (8) mM as shown in Figs. 7A2, B2.
Fig. 7: Serratia bacteria: (A) Dilution 10-3 without AgNPs. (A1) Dilution 10-3 with AgNPs of 6mM.(A2) Dilution 10-3 with AgNPs of 8mM. (B) Dilution 10-4 without AgNPs. (B1) Dilution 10-4 with AgNPs of 6mM. (B2) Dilution 10-4with AgNPs of 8mM
Fig. 8 shows Staphylococcus bacterial before and after treatment by AgNpsin different concentrations (6 and 8) mM. The colonies are grown without treatment at dilutions 10-3 and 10-4 as shown in Figs. 8A, B. The colonies are not detected, when treated with AgNPs concentration (6) mM at dilutions 10-3 and 10-4 as shown in Figs. 8A1, B1. The colonies are not detected with AgNPs concentration (8) mM as shown in Figs. 8A2, B2.
Fig. 8 Staphylococcus bacterial:(A) without AgNPs. (B) Treatment by AgNPs (6) mM. (C) Treatment by AgNPs (8) mM
Discussion
When bacteria are treatment with AgNps of (6) mM found 7 Proteus colonies at dilution of 10-3this means that the concentration (8) mM better than 6 mM in process for bacterial activity. Highest number of colonies obtained in pseudomonas type at dilution (10-3) without AgNPs. Whereas, the lowest number is obtained of Proteus at dilution (10-3) and concentration (6 mM). The colonies are grown without treatment at dilutions 10-3and 10-4as shown in Figs. 2A, B. The colonies are not detected, when treated with AgNPs concentration (6) mM at dilutions 10-3and 10-4as shown in Figs. 2A1, B1. The colonies are not detected with AgNPs concentration (8) mM as shown in Figs. 2A2, B2.The colonies are grown without treatment at dilutions 10-3 and 10-4 as shown in Figs. 3 A, B. whereas, the low number (7 colonies) is obtained of Proteus at dilution (10-3) with concentration (6 mM) as shown in Fig. 2 A1. The colonies are not detected, when treated with AgNPs concentration (6) mM at dilutions 10-4 as shown in Fig. 3 B1. The colonies are not detected with AgNPs concentration (8) mM as shown in Figs. 3 A2, B2.The colonies are grown without treatment at dilutions 10-3and 10-4as shown in Figs. 4A, B. The colonies are not detected, when treated with AgNPs concentration (6) mM at dilutions 10-3and 10-4as shown in Figs. 4A1, B1. The colonies are not detected with AgNPs concentration (8) mM as shown in Figs. 4A2, B2.The colonies are grown without treatment at dilutions 10-3and 10-4as shown in Figs. 5A, B. The colonies are not detected, when treated with AgNPs concentration (6) mMat dilutions 10-3and 10-4as shown in Figs. 5A1, B1. The colonies are not detected with AgNPs concentration (8) mM as shown in Figs. 5A2, B2.The colonies are grown without treatment at dilutions 10-3and 10-4as shown in Figs. 6A, B. The colonies are not detected, when treated with AgNPs concentration (6) mM at dilutions 10-3and 10-4as shown in
Figs. 6A1, B1. The colonies are not detected with AgNPs concentration (8) mM as shown in Figs. 6A2, B2.The colonies are grown without treatment at dilutions 10-3and 10-4as shown in Figs. 7A, B. The colonies are not detected, when treated with AgNPs concentration (6) mM at dilutions 10-3and 10-4as shown in Figs. 7A1, B1. The colonies are not detected with AgNPs concentration (8) mM as shown in Figs. 7A2, B2.The colonies are grown without treatment at dilutions 10-3 and 10-4 as shown in Figs. 8A, B. The colonies are not detected, when treated with AgNPs concentration (6) mM at dilutions 10-3 and 10-4 as shown in Figs. 8A1, B1. The colonies are not detected with AgNPs concentration (8) mM as shown in Figs. 8A2, B2.
Conclusions
AgNps are affected on growth most bacterial pathogens activity. The results showed no growing bacterial after added AgNps with huge active at concentration (8) mM.It concluded that AgNps with concentrations (6 and 8 mM) can be used as antibacterial. The silver nano-particles are reported to inhibit the growth of pathogenic bacteria (E.coli, klebsiella, bacillus, pseudomonas, Proteus, Serratia, staphylococcus).
Acknowledgment
The author acknowledges the financial support of the College of Science of the University of Kufa.
References
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