الفهرس 
INTRODUCTIONOnly 14 pages are availabe for public view
 |  | from | 216 |  |  |
| | | from | 216 | | |
Abstract
In the current study, a total of 25 bacterial isolates were isolated from soil samples. The bacterial isolates were screened for their ability and efficiency to produce silver nanoparticles by extracellular synthesis mechanisms. This was observed by the change in the color of its supernatant from yellow to dark brown when treated with AgNO3. Only one of the 25 tested isolates, coded as S3, was found to have the ability to synthesize AgNPs. The production of AgNPs using S3 supernatant has been confirmed by UV–vis spectroscopy as part of the primary confirmation. The obtained absorption showed a characteristic peak for AgNPs at 420 nm. The isolate S3 was chosen as the successful candidate for the biosynthesis of AgNPs and was used throughout the work after its submission for molecular identification by a 16S rRNA sequencing-based method. The 16S rRNA sequence of the strain S3 was found to be phylogenetically similar to those of Leclercia adecarboxylata, and hence the S3 isolate was named Leclercia adecarboxylata THHM. The sequenced gene was submitted to GenBank with the accession number OK605882. Leclercia adecarboxylata is a Gram-negative motile rod-shaped one that is belonging to the genus Leclercia of the family Enterobacteriaceae. To the best of our knowledge, this study reports for the first time the biosynthesis of AgNPs using the supernatant of Leclercia adecarboxylata.
Through one-variable-at-a-time (OVAT) analysis, various parameters, including incubation time, AgNO3 concentration, pH, temperature, and bacterial supernatant concentration, were optimized for the biosynthesis of AgNPs using the supernatant of Leclercia adecarboxylata THHM. Using the supernatant of Leclercia adecarboxylata THHM, the optimal parameters for enhancing the biosynthesis of AgNPs using OVAT analysis were an incubation time of 48.0 h, a concentration of silver nitrate of 1.0 mM, a temperature of 40.0 °C, a pH of 7.0, and a supernatant concentration of 20% (v/v). The variables, which significantly affected the biosynthesis of AgNPs, were determined statistically using the Plackett-Burman design (PBD). This design was used to define the most optimal levels of factors affecting the biosynthesis of AgNPs using the supernatant of Leclercia adecarboxylata THHM. The five independent variables selected were the AgNO3 concentration, bacterial supernatant concentration, incubation time, pH, and illumination. According to the PBD, the long incubation time (72.0 h), the high AgNO3 concentration (1.5 mM), the high bacterial supernatant concentration (30%), and the presence of light had a positive effect on the biosynthesis of AgNPs. On the other hand, the lower pH level (7.0) resulted in high AgNPs biosynthesis. The most significant factors affecting the biosynthesis of AgNPs were illumination, followed by AgNO3 concentration, pH, bacterial supernatant concentration, and incubation time. The overall optimal parameters for enhancing the biosynthesis of AgNPs, which were used in the final production of AgNPs, were an incubation time of 72.0 h, a concentration of 1.5 mM silver nitrate, a pH of 7.0, and a supernatant concentration of 30% (v/v) under illumination conditions at a temperature of 40.0 °C.
AgNPs were also chemically synthesized by reducing AgNO3 with glucose, to be compared with those produced biologically. The biosynthesized AgNPs (bio-AgNPs) and chemically synthesized AgNPs (chem-AgNPs) were characterized using several advanced analytical techniques, including UV-visible spectroscopy, TEM, and FTIR spectroscopy. The UV-vis absorption spectra of bio-AgNPs (under optimized conditions using the supernatant of Leclercia adecarboxylata THHM) and chem-AgNPs showed characteristic peaks for AgNPs at 423 and 420 nm, respectively. The TEM images of bio- and chem-AgNPs showed spherical nanoparticles with uniform dispersion. The bio-AgNPs were smaller, ranging in size from 3.48 to 39.02 nm and having an average particle size of 17.43 nm, whereas the chem-AgNPs were relatively larger, ranging in size from 13.08 to 53.71 nm and having an average particle size of 35.06 nm. In general, no substantial variations were found in the optical spectra recorded by bio-AgNPs and chem-AgNPs, particularly in regards to the color, shape, and position of the absorption bands. HRTEM micrographs with magnified lattice fringes revealed the crystalline nature of the bio- and chem-AgNPs. The FTIR analysis was conducted to identify the possible biomolecules that are responsible for reducing, capping, and stabilizing silver nanoparticles. The FTIR spectra of the bio-AgNPs showed three distinct peaks at 3321.50, 2160.15, and 1636.33 cm-1, which are attributed to the primary amines and hydroxyl groups stretching vibrations in peptide linkages, alkynes groups stretching vibrations, and carbonyl stretch vibrations in the amide linkages of proteins, respectively. The FTIR spectra of the chem-AgNPs showed absorption peaks at 3321.47, 2133.48, and 1636.56 cm-1. The FTIR spectra of chem-AgNPs and bio-AgNPs were highly similar. The functional groups involved in the chemical reduction of silver ions into AgNPs, as well as their stability, were comparable to those previously described in biosynthesized AgNPs.
The antimicrobial activity of the bio-AgNPs was compared with that of the chem-AgNPs against clinically important microbial pathogens using the disc diffusion assay. In addition, both bio- and chem-AgNPs have been used as comparable antimicrobial agents with various antibiotics as standards. The microbial pathogens used in the assay were Staphylococcus aureus ATCC6538, Bacillus cereus ATCC6633, Escherichia coli NCTC10418, Vibrio cholerae ATCC700, Pseudomonas aeruginosa ATCC9027, Klebsiella pneumoniae ATCC13883, and Candida albicans ATCC700. In general, bio-AgNPs showed higher antimicrobial activity than chem-AgNPs against all tested pathogenic strains. Vibrio cholerae ATCC700 strain was the most affected bacterial pathogen, whereas Pseudomonas aeruginosa ATCC9027 strain was the least affected. The bio-AgNPs’ pattern of the antimicrobial activity against the tested pathogens was as follows: Vibrio cholerae ATCC700 > Staphylococcus aureus ATCC6538 = Klebsiella pneumoniae ATCC13883 > Bacillus cereus ATCC6633 = Escherichia coli NCTC10418 = Candida albicans ATCC700 > Pseudomonas aeruginosa ATCC9027. The chem-AgNPs’ pattern of the antimicrobial activity against the investigated pathogens was as follows: Bacillus cereus ATCC6633 > Staphylococcus aureus ATCC6538 = Vibrio cholerae ATCC700 = Candida albicans ATCC700 > Escherichia coli NCTC10418 = Klebsiella pneumoniae ATCC13883 > Pseudomonas aeruginosa ATCC9027. There was a considerable variation in the antimicrobial activities of the various types of antibiotics used against the microbial pathogens. Ampicillin (10 µg) had the highest antimicrobial activity against all test strains, followed by tetracycline (30 µg), ciprofloxacin (5 µg), vancomycin (30 µg), gentamicin (10 µg), and ceftriaxone (30 µg).
The antimicrobial activity and potency of AgNPs have been quantitatively assessed by determining the minimum inhibitory concentration (MIC) values. The tested microbial pathogens were separately exposed to bio- and chem-AgNPs at concentrations ranging from 2000 μg/ml to 3.9 μg/ml. All microbial pathogens were found to be more susceptible to bio-AgNPs than chem-AgNPs, with MIC values of 500 to 1000 µg/ml and 1000 to 2000 µg/ml for bio-AgNPs and chem-AgNPs, respectively. These MIC values were determined using a resazurin-based microtiter dilution assay. All the microbial pathogens have the same MIC value of 500 µg/ml with bio-AgNPs except for Klebsiella pneumoniae ATCC13883, which has a higher MIC value of 1000 µg/ml. The lowest MIC for chem-AgNPs was 1000 µg/ml for Vibrio cholerae ATCC700, whereas the remaining microbial pathogens had the same higher MIC of 2000 µg/ml.
The nanocomposites with antimicrobial properties that incorporate AgNPs with nanofibers have potential applications in various fields. The nanofibers were successfully prepared from the polymeric solutions AgNPs-free PVA/PEO/β-CD, PVA/PEO/β-CD/bio-, and chem-AgNPs using the electrospinning technique. The field emission scanning electron microscopy (FE-SEM) micrographs of the composite nanofiber membranes showed that they were well formed and had long, smooth, uniform, and bead-free nanofibers for all electrospun samples. The composite nanofibers had a homogeneous nanostructure and no distinct phase separation, confirming the compatibility and miscibility of the components in the composite nanofibers. The AgNPs were clearly identified in nanofiber composites containing both bio- and chem-AgNPs, indicating that AgNPs were successfully integrated into PVA/PEO/β-CD nanofibers. The average fiber diameter within the nanofibrous membrane of the AgNPs-free PVA/PEO/β-CD was 171.5 nm. When PVA/PEO/β-CD was incorporated with bio- and chem-AgNPs, there was no obvious change in the nanostructure of the nanofibers. The average fiber diameters varied slightly for the nanofiber composites PVA/PEO/β-CD/bio-AgNPs and PVA/PEO/β-CD/chem-AgNPs, which were 204.3 and 150.5 nm, respectively. FTIR spectroscopy was used to characterize the functional groups of PVA/PEO/β-CD/bio- and chem-AgNPs nanofiber composites. The vibration bands of the functional groups of the PVA/PEO/β-CD nanofiber composites confirmed the presence of the polymer PVA/PEO/β-CD components.
The antimicrobial activity of the polymeric PVA/PEO/β-CD/bio- and chem-AgNPs nanofiber composites against the tested microbial pathogens was studied by measuring the inhibition zone by the disc diffusion assay. All PVA/PEO/β-CD nanofiber composite samples containing AgNPs have good inhibitory activities against the tested microbial pathogens. The PVA/PEO/β-CD/bio-AgNPs nanofiber composite exhibit higher antimicrobial activity against the tested microbial pathogens than the PVA/PEO/β-CD/chem-AgNPs nanofiber composite. This confirmed previous findings, which showed that bio-AgNPs have higher antimicrobial activity compared to chem-AgNPs against the tested pathogenic strains. The most affected bacterial pathogen was Vibrio cholerae ATCC700 strain, which was highly affected by PVA/PEO/β-CD/bio-AgNPs nanofiber composite compared to PVA/PEO/β-CD/chem-AgNPs nanofiber composite. The Klebsiella pneumoniae ATCC13883 strain is the bacterial pathogen least affected by both PVA/PEO/β-CD/bio- and chem-AgNPs nanofiber composites.
CONCLUSION
Twenty-five bacterial isolates were isolated from soil samples. The ability of the isolated bacteria to synthesize AgNPs was screened by the color change. The isolated strain that showed the capability of reducing Ag ions into AgNPs with the formation of a characteristic AgNPs peak at 420 nm was selected and submitted for molecular identification. The sequenced 16S rRNA gene was submitted to GenBank with the accession number OK605882.
The optimal parameters for enhancing the biosynthesis of AgNPs using the supernatant of Leclercia adecarboxylata THHM were optimized through OVAT analysis and statistically using the PBD. The overall optimum parameters, which were used in the final production of AgNPs, were an incubation time of 72.0 h, a concentration of 1.5 mM silver nitrate, a pH of 7.0, and a supernatant concentration of 30% (v/v) under illumination conditions at a temperature of 40.0 °C.
The bio- and chemically synthesized AgNPs were characterized by UV-visible spectroscopy, TEM, and FTIR. The UV-vis spectra of bio-AgNPs under optimized conditions and chem-AgNPs showed characteristic AgNPs peaks at 423 and 420 nm, respectively. Both bio- and chem-AgNPs had spherical nanoparticles. The bio-AgNPs were smaller than the chem-AgNPs, with an average particle size of 17.43 and 35.06 nm, respectively. The FTIR spectra of the bio- and chem-AgNPs revealed similar peaks. The stability of AgNPs and the functional groups used in their bio- and chemical production were comparable.
The bio- and chem-AgNPs were found to be effective against important clinical pathogens. The bio-AgNPs showed higher antimicrobial activity than chem-AgNPs against all tested pathogenic strains, with MIC values of 500 µg/mL for all microbial pathogens except for Klebsiella pneumoniae ATCC13883, which has 1000 µg/ml. The lowest MIC for chem-AgNPs was 1000 µg/ml for Vibrio cholerae ATCC700, while the other tested microbial pathogens had MIC values of 2000 µg/ml.
Nanocomposites with antimicrobial properties that incorporate AgNPs with nanofibers have potential applications in various fields. Nanofiber-nanosilver polymeric composites for different antimicrobial applications have been successfully prepared with no clear phase separation and homogeneous nanostructure. Both bio- and chem-AgNPs were successfully integrated into PVA/PEO/β-CD nanofibers with long, smooth, uniform, bead-free, and well-formed nanofibers for all electrospun polymeric samples.
The antimicrobial activity of the polymeric PVA/PEO/β-CD/bio- and chem-AgNPs nanofiber composites against the tested microbial pathogens was studied by measuring the inhibition zone by the disc diffusion assay. The PVA/PEO/β-CD/bio-AgNPs nanofibers composite exhibit higher antimicrobial activity against the microbial pathogens than the PVA/PEO/β-CD/chem-AgNPs nanofibers composite.
These results indicated for the first time the ability of the supernatant of Leclercia adecarboxylata THHM under optimum conditions to biosynthesize small-size AgNPs that have acceptable antimicrobial activity compared to chem-AgNPs against important clinical pathogens. The polymeric PVA/PEO/β-CD/bio- and chem-AgNPs nanofiber composites can be used in a wide range of applications in various fields due to their antimicrobial activity.