Antimicrobial Activity of Silver Nanoparticles on Pathogenic Bacteria

Nosocomial infection is acquired contamination of hospitals and health care units caused by multidrug resistant bacteria. Currently, bacterial resistance to antimicrobial medication represents a complicated public health problem. Recent studies on the antimicrobial activity of silver nanoparticles (AgNPs) attracted researchers worldwide to focus on the safe synthesis of AgNPs as antimicrobial agents against multidrug resistant bacteria. The antimicrobial efficacy of AgNPs on pathogenic bacteria isolated from clinical cases of acquired hospital infection was targeted in this project. Fifty specimens of stool were collected through private laboratories in Baghdad from patients who suffered diarrheal symptoms. Bacterial isolation, identification, and characterization via culturing on MacConkey agar, Salmonella shigella agar, and IMVic analysis were done besides, using polymerase chain reaction (PCR) through amplifying inf B gene for molecular characterization. The obtained isolates were tested for antimicrobial sensitivity via disk diffusion assay against; Gentamycin, Amoxicillin, Tetracycline, Ceftriaxone and a suspension of silver nanoparticles (1mM AgNo3 reduced by 1% tri-sodium citrate). Results of isolation and IMVic showed the obtained isolates were Klebsiella spp., Enterobacter spp., Citrobacter spp., and PCR assay confirmed their pathogenicity. Disc diffusion assay showed the sensitivity of the isolates (mm); Gentamycin (24.94 ± 0.1), Amoxicillin (2.11 ± 0.13), Tetracycline (12.15 ± 0.1), Ceftriaxone (12.35 ± 0.1). Whereas, all isolates are sensitive to AgNPs (24.12 ± 0.3). This result of the antimicrobial effect of AgNPs on nosocomial infection promises for developing AgNPs solution as a product used in the sterilization of furniture, floors and hospital water cycles.


Introduction
Nosocomial infections comprise infections obtained from the hospital or other health care canters, which appear for the first time within 48 hours of admission to the hospital, three days after surgery or 30 days after the surgical operation.Hospital infections had become a major health challenge around the world in the latest decades.Owing to the increase in the number of hospitals and health centres besides resistance against antimicrobial medications and render some of them ineffective after only a few years of their application besides, trade of different types of meat and dairy products 2 .Silver has long been used as an antimicrobial agent in wound healing, both in its solid state and with saline solutions for healthy wounds.Nowadays, dressings saturated with AgNO3 3 can be found, silver shows very interesting properties due to its chemical stability, good conductivity, catalytic activity, and antibacterial.Moreover, AgNPs silver nanoparticles are one of the most widely studied particles at present 4 , and AgNPs have been applied in various fields such as textile, cosmetics, food industry and biomedicine.In the field of biomedicine, AgNPs gaining particular strength due to their applications antimicrobial agents, coatings for medical devices, and as carriers for chemotherapy drugs 5 .Although AgNPs have been extensively studied, there is still a need for ongoing research toward developing more biologically sustainable synthesis methodologies, along with uncovering the mechanisms involved in toxicology 6 .
Three categories of techniques have been followed in previous research for the preparation of AgNPs [7][8] .Chemical procedures based on the reduction of silver ions in organic or aqueous solution, chemical and physical processes (electrochemical and photophysical) and biosynthesis (by plants).For chemical methods in aqueous solution, various reducing agents have been suggested, such as sodium citrate, sodium borohydride, hydrazine, glucose, ascorbic acid, gallic acid, and various plant extracts 6,9 .Of the range of chemical methods available for production, chemical reduction is the most commonly used of this type of nanosystem.Chemical synthesis of AgNPs is easy to apply on a large scale and low-priced 4 .AgNPs exhibit effective antibacterial properties against both Gram-positive and Gram-negative bacteria, as well against methicillin-resistant strains 10 .Besides, AgNPs display anti-biofilm activities 11, and have synergistic action with different types of antibiotics eg, b-lactams, macrolides, and lincosamide 12 .Gram-positive and Gram negative bacteria are significant causes of different infections, principally in hospitals, and are resistant to many antimicrobial agents.Due to the prevalence and rise in bacterial resistance to antibacterial medications, many scientists have been interested in developing antimicrobial agents that are resistance-free and low cost 13 .Such challenges and necessities had led to the use of silver nanoparticles as antiseptics and broadspectrum activity and far lower susceptibility to induce microbial resistance 14 .Therefore, this study aimed to prepare products of silver nanoparticles; effective against bacterial contamination, ecofriendly, and at low prices for hospital hygiene.

Methodology
Specimen's collection: 50 samples of stool (clinical cases of diarrhoea) were collected from privet laboratories in Baghdad in septic conditions and transported within one hour to the laboratory of bacteriology at Ibn Sina University.Cultured media and chemicals: all media and biochemical used for the isolation and identification of bacteria were purchased from Hi Media/ India.Isolation of bacteria: 10g of each stool specimen was inoculated in 90ml flask of sterile peptone water and incubated at 37ᴼC for 24-48hrs.Then, all inoculated flasks were subjected to test for confirmation of bacterial growth.Each flask that was positive for bacterial growth was streaked on MacConkey agar (MAC) and Salmonella Shigella agar (SSA) after preparation of serial dilution of bacteria in normal saline 15.Identification by IMViC: biochemical identification was done through IMViC test; for each obtained isolate, sterile slants of Indole, Methyl red, Voges-Proskauer and citrate, were stabbed with fresh bacterial inoculum 5x105 then, incubated at 37ᴼC for 24-48 hrs 10 .Molecular detection: was performed through DNA extraction, PCR amplification and gel electrophoresis.Genomic DNA was obtained according to Wizard genomic DNA purification kit (Promega).A fresh subculture 5x10 5 cell /ml of fresh inoculum was prepared for each recovered isolates and incubated at 37ᴼC. Isolation of genomic DNA from gram negative bacteria was done according to manufacturer instruction as follow: first, cell pellet was prepared by centrifuging 1ml of culture for 2 min at 13,000 -16,000 xg, then the supernatant was discarded.A total of 600µl of nuclei lysis solution was added to the cell pellet with gently pipetting to mix well and incubated for 5 min at 80ᴼC then, cooled to room temperature.About 3 µl of RNase solution was added mixed and incubated for 15-60 min at 37ᴼC following the PCR protocol: Initial denaturation starting at 95°C for 5mim then, 1min.Annealing temperate ranging 68°C for 90sec.Initial extension at 72°C for 90s followed by final extension for 5min.
The PCR products were seen by UV after electrophoresing on 1.5% agarose gel/100volt.Both forward and reverse primers, Table .1 were designed by 16 Chemical synthesis of nanoparticles: Synthesis of silver nanoparticles was done under constant heating through reducing AgNo3 (starting agent) by trisodium citrate C6H5O7Na3.17.0mg of silver nitrate was added to 250 ml deionised water to prepare 1mM of AgNO3 solution and adjusted in a microwave oven for constant heating to let the solution dissolves absolutely.10 ml (1%) solution of tri-sodium citrate was added to AgNO3 solution drop by drop with mixing vigorously.The mixture was heated and the colourless solution changed to yellowish brown.The solution was the removed from microwave oven and still at room temperature with stirring to cool at 28ᴼC 17 .
Antimicrobial sensitivity: to evaluate the efficacy of AgNO3 solution as an antimicrobial agent; discs were prepared from filter paper and autoclaved, then dispense in the AgNO3 solution until soaked all the solution and dried.At the same time antibiotic sensitivity test was performed in the disc diffusion technique.Moller Hinton plates were prepared and streaked with 0.5 MacFerland of a fresh culture of each isolate/double.Then, the plates were dispensed with antibiotic discs; Gentamycin, Amoxicillin, Tetracycline and Ceftriaxone.Besides AgNPs dried filter pares were dispensed on Moller Hinton plates also streaked with 0.5 MacFerland of a fresh culture of each isolate/double.

Results
Isolation outcomes showed; out of 50 stool cultured specimens, 26 isolates were positive to grow on MacConkey agar (the differential media to lactose ferment bacteria of Enterobacteriaceae), and pink colonies due to lactose ferment were seen.Whereas, none of the isolates were positive to grow on SSA with gas production,  Chemical synthesis of silver nanoparticles showed the dark brown colouration of reduced solution of AgNO3 after continuous heating for 15min and above.

Table3. Results of antimicrobial sensitivity assay and antimicrobial activity of silver nano particles on clinical bacterial isolates.
a,b Statistical significant difference (P<0.05),a>b *Non-significant.

Discussion
Results of this study showed the antimicrobial efficacy of AgNPs against pathogenic bacteria of nosocomial infection.Globally, health care associated infections have been grown into a public health concern -18 .The nosocomial infection ranges in developed and developing countries between 7 to10%.This variation in antimicrobial sensitivity refers to many factors such as; the prevalence of resistant pathogens, ill-use of antibiotics, the severity of the infection and a long stay in hospital.It's very necessary to control nosocomial infection since it becomes one of the death cause among hospital staying patients in recent years ‫و‬ .Outcomes of this study revealed the  11,20 .The three major sites for nosocomial infection include the urinary tract system (31%), the respiratory system (24%), and the bloodstream (16%).Nosocomial infection can also occur in the skin and other organs.Pneumonia, urinary tract infection, and septicaemia are the most commonly diagnosed nosocomial infection on the three major sites.It has been reported that Ventilator-associated Pneumonia (VAP) is the most common nosocomial infection in the intensive care unit (ICU) and responsible for approximately fifty percent of all hospital-acquired pneumonia cases and UTI is the most common NIs in the developed countries. 21hemical mediated synthesis SNPs and their efficacy as antibacterial against Gram negative bacteria were described by 1 .They proposed that SNPs penetrate cell wall of G-bacteria and attach to the cell membrane at surface and releasing Ag ions that disturb its function as well 9,21 were suggested the same to antibacterial activity against gram positive bacteria , 22,23 .

Conclusion
The present study is principally concerned with chemically mediated synthesis of silver nanoparticles through reducing of silver with trisodium citrate and obtained silver nanoparticles.Besides, the efficacy of SNPs on G-bacteria isolated from clinical cases of hospital acquired infection was approved.Therefore, the promising results encourage to the development of SNPs products to use in the hygiene of hospitals.Furthermore, studies on the efficacy of SNPs on G+ bacteria are required to enhance the need for SNPs products as detergents.-Authors sign on ethical consideration's approval.

Author's Declaration
-Ethical Clearance: The project was approved by the local ethical committee in the Iraqi Ministry of Science and Technology.
2024, 21(3): 0937-0943 https://dx.doi.org/10.21123/bsj.2023.8728P-ISSN: 2078-8665 -E-ISSN: 2411-7986 Baghdad Science Journal 2024, 21(3): 0937-0943 https://dx.doi.org/10.21123/bsj.2023.8728P-ISSN: 2078-8665 -E-ISSN: 2411-7986 Baghdad Science Journalthen, cooled to room temperature.A total of 200µl of protein precipitation solution was added and vortex for cell precipitation and the sample was incubated on ice for 5min.After that the sample was centrifuged for 3 min at 13,000-16,000 xg.About 800µl of supernatant was transferred to a clean tube containing 600µl of 99% Isopropanol at room temperature with inverting the tube until the white thread of DNA appeared.The samples were centrifuged for 2 min at 13,000-16,000 xg and the supernatant was discarded for DNA precipitation and rehydration.About 600µl of 70 % ethanol was added and mixed by upside down inverting then, centrifuged for 2 min at 13,000 -16,000 xg.Ethanol was aspirated and the pellet was air dried for 10 -15 min.A total of 100µl of rehydration solution was added to rehydrate the DNA pellet for 1 h at 65 ᴼC or the pellet could be kept overnight at 4ᴼC.Then, DNA was electrophoresed on 7% agarose gel and detected by UV gel doc device.All positive samples of DNA are stored at -20ᴼC until use.DNA amplification: All reactions were prepared for all isolates in a final volume 25µl/0.5mlPCR tube.Each reaction was contained (Master Mix 12.5µl, F primer-1.25µl,R primer-1.25µl,free RNA water 5µl and DNA template 5µl in addition to control).

-
Conflicts of Interest: None.-We hereby confirm that all the Figures and Tables in the manuscript are ours.Furthermore, any Figures and images, that are not ours, have been included with the necessary permission for republication, which is attached to the manuscript.

Table 2 . Results of IMViC analysis and PCR assay for characterization and identification of collected isolates.
/dx.doi.org/10.21123/bsj.2023.8728P-ISSN: 2078-8665 -E-ISSN: 2411-7986 Baghdad Science Journal prevalence of gram-negative, lactose ferment, bacteria 52% among pathogens associated with hospital infection is too high, and agreed with the results of Tolera M et al, 19 .Besides, results of this study reported Citrobacter spp., Enterobacter spp.and Klebsiella spp., concordant with outcomes obtained by