Biogenic Synthesis, Characterization and Antibacterial Properties of Silver Nanoparticles against Human Pathogens

activi-Abstract: Biogenic synthesis of silver nanoparticles (AgNPs) is more eco-friendly and cost-effective approach as compared to the conventional chemical synthesis. Biologically synthesized AgNPs have been proved as therapeutically effective and valuable compounds. In this study, the four bacterial strains Escherichia coli (MT448673), Pseudomonas aeruginosa (MN900691), Bacillus subtilis (MN900684) and Bacillus licheniformis (MN900686) were used for the biogenic synthesis of AgNPs. Agar well diffusion assay revealed to determine the antibacterial activity of all biogenically synthesized AGNPs showed that P. aeruginosa AgNPs possessed significantly high ( p < 0.05) antibacterial potential against all tested isolates. The one-way ANOVA test showed that that P. aeruginosa AgNPs showed significantly ( p < 0.05) larger zones of inhibition (ZOI: 19 to 22 mm) compared to the positive control (rifampicin: 50 µg/mL) while no ZOI was observed against negative control (Dimethyl sulfoxide: DMSO). Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) concentration against four test strains also showed that among all biogenically synthesized NPs, P. aeruginosa AgNPs showed effective MIC (3.3-3.6 µg/mL) and MBC (4.3-4.6 µg/mL). Hence, P. aeruginosa AGNPs were characterized using visual UV vis-spectroscopy, X-ray diffractometer (XRD), fourier transform infrared (FTIR) and scanning electron microscopy (SEM). The formation of peak around 430 nm indicated the formation of AgNPs while the FTIR confirmed the involvement of biological molecules in the formation of nanoparticles (NPs). SEM revealed that the NPs were of approximately 40 nm. Overall, this study suggested that the biogenically synthesized nanoparticles could be utilized as effective antimicrobial agents for effective disease control.

2 ties 4, 5 and emerging as promising nano antibiotics now a days 6 . Because of their broad-spectrum activities, AgNPs grab great attention in the field of biomedical, medicine, agriculture, insect control and several other industries 7 . By using an ecofriendly approach, AgNPs applications have been increased in preparation of large number of products such as pests, electronic devices and in controlling microbe s growth and infection 8,9 . Silver nanoparticles AgNPs are most commercialized among inorganic nanoparticles. They are nontoxic, safe inorganic antibacterial agent used for centuries and has great potential to kill disease causing microorganisms.
The use of silver as suspension and in nano-particulate form has a dramatic revival in nanotechnology. It has eminence antibacterial potency against human pathogens. The main task in nanoparticles synthesis is the control of their physical properties like uniform particle size, similar shape, chemical composition, morphology and crystal structure. The reduced effectiveness of drugs, antibiotics against the pathogenic bacteria, particularly against multi-drug resistance MDR bacteria could be the major threat to the life of the human being 1,10 . AgNPs have very strong antibacterial activity against the human pathogens, because of their effectiveness against most of the microbial pathogens, microorganisms have been probed as potential bio-factories for metallic nanoparticles synthesis such as silver, copper, zinc and gold biogenic synthesis 11 .
Moreover, the antibacterial activity of biogenic nanoparticles in combination with antibiotics signifies their importance in combating multi-drug resistant MDR pathogenic bacteria in planktonic 12 and biofilm mode microorganisms embedded in elf produced polymeric matrix 13 . It was also observed that drug-loaded silver nanoparticles Ciprofloxacin 10 mM showed a stronger antibacterial potential than the synthesized silver nanoparticles and ciprofloxacin alone to restrict the development of E. coli and E. aerogenes 14 . Biogenically synthesized nanoparticles are easy to produce biocompatible, economic, environmental friendly and offer different catalytic abilities compared to chemically synthesized ones. They have anticancer and antioxidant properties. Furthermore, they are naturally stabilized, as the natural organic material citrate, sodium dodecyl sulfate of bacteria work as natural capping layers surrounding the biogenic nanoparticles, make these active, stable and reusable 1,12,15,16 . These act as promising antimicrobial agent due to their long term stability and biocompatibility 17 .
The application of biogenic AgNPs has been demonstrated since it arrests the growth and multiplication of many bacteria such as Bacillus cereus, B. subtilis, Staphylococcus aureus, Citrobacter koseri, Salmonella typhii, P. aeruginosa, E. coli, Klebsiella pneumoniae, and Candida albicans by binding Ag/Ag with the biomolecules present in the microbial cells. It has been suggested that biogenic AgNPs produce reactive oxygen species and free radicals which cause cell death through apoptosis and prevent their replication. Since AgNPs are smaller than the microorganisms, they diffuse into cell and rupture the cell wall which has been shown from SEM. It has also been observed that smaller nanoparticles are more effective than the bigger ones 1,18 .
In this study, microbial synthesis of AgNPs was investigated by using culture supernatant of the bacterial strains E. coli MT448673 , P. aeruginosa MN900691 , B. subtilis MN900684 and B. licheniformis MN900686 , as a reducing agent. The biogenically synthesized AgNPs was further characterized for the antibacterial potency against four human pathogenic bacteria both Gram negative and Gram positive using agar well diffusion method. The P. aeruginosa AgNPs showed significant antibacterial potential, hence were further characterized by UV-vis spectroscopy, X ray diffraction XRD , scanning electron microscopy SEM and fourier transform infrared spectroscopy FTIR .

Test microorganisms
Four human pathogenic bacteria both Gram negative and Gram positive such as E. coli MT448673 , P. aeruginosa MN900691 , B. subtilis MN900684 and B. licheniformis MN900686 were obtained from Microbiology lab, Government College University, Lahore and used in the current study.

Chemicals and reagents
The chemicals, media, reagents and AgNO 3 used during experiments were highly pure and up to the analytical grade. The chemicals including silver nitrate Merck, Germany , Luria bertani LB agar and LB broth were purchased from Sigma-Aldrich, USA.

Preparation of supernatant
Luria bertani LB broth was prepared by dissolving 5.0 g Sodium chloride, 5.0 g peptone; 3.0 g yeast extract in 500 mL distilled water, which after adjusting pH was made up to 1 liter. The prepared LB was sterilized by autoclaving at 121 for 15 minutes at 15 Ib and inoculated with 100 µL OD: 1 0.2 fresh cultures of E. coli, P. aeruginosa, B. subtilis and B. licheniformis, separately. The culture flasks were incubated for 24 h in incubator at 37 at a rotatory shaker 2g-force . Following incubation, the bacterial cultures were centrifuged at 10000 rpm for 10 min twice. The supernatants were saved for further study.

Biosynthesis of silver nanoparticles
For synthesis of AgNPs, each supernatant was mixed with 10 mM solution of AgNO 3 , in 2:1 supernatant: AgNO 3 3 solution while two control flasks, each with AgNO 3 10 mM and bacterial culture separately were run in parallel. The prepared solutions were incubated for 72 h on rotatory shaker at 2g-force at 37 . All the solutions were kept in dark to prevent photochemical reaction during the experiment. After three days, flasks having supernatants and AgNO 3 turned showed color change from yellow solution to brown confirming synthesis of AgNPs. No colour change was observed in control flask. The cultures were centrifuged at 3g-force for 10 minutes, the pellet was discarded and the supernatant was saved for further characterization.

Antibacterial activity of AgNPs
The antibacterial activity of AgNPs was analyzed through agar well diffusion method against the test pathogens i.e., E. coli, P. aeruginosa, B. subtilis and B. licheniformis 19 22 . The Muller Hinton agar plates were prepared and well of 6 mm diameter were made using sterile cork borer. The test pathogens culture was adjusted to 0.5 McFarland turbidity standard and was spread on the media plate uniformly. The 100 µL of AgNPs 1 mg/mL was added into each well. DMSO, rifampicin 50 µg/mL and AgNO 3 were used as controls. The plates were kept at room temperature for one hour. Afterwards, plates were incubated at 37 for 24 h. Following incubation period, the clear zones around the wells were taken as zone of inhibition ZOI and were recorded in millimeter mm .

Determination of minimum inhibitory concentration
MIC and minimum bactericidal concentration MBC MIC and MBC of AgNPs were measured using tube dilution method following Liaqat et al. 23 with slight modifications. In brief, 3 mL of freshly prepared nutrient broth was added into the test tubes. About 10 µL of bacterial culture which adjusted to 0.5 McFarland turbidity standard was added to the tubes containing broth. The test tubes were supplied with various concentrations 3-12 µg/mL of AgNPs and incubated at 37 for 24 h. Controls having AgNO 3 and LB medium were run in parallel. Following incubation, MIC was measured by considering the lowest possible concentration that inhibited the bacterial growth visually, while the MBC was determined by spreading the lowest MIC on media plates which killed 99.9 of bacteria.

Characterization of P. aeruginosa AgNPs
P. aeruginosa synthesized AgNPs showed excellent antibacterial potential hence subjected to the further characterization via UV-Visible spectroscopy, FTIR, SEM, and XRD in order to confirm the formation and specificity size, shape and peak of AgNPs.

UV-Visible spectroscopy
The P. aeruginosa AgNPs supernatant was qualitatively analyzed by UV-visible spectroscopy using AE-S70-1U UVvisible spectrophotometer and AgNO 3 solution was used as control. UV-vis spectrophotometer from 300 to 770 nm operated at a resolution of 1 nm was used as a function of wavelength for spectral analysis of AgNPs. Occurrence of peak between 400-470 nm showed formation of AgNPs and reduction of silver nitrate.

Fourier transform-infrared FTIR spectroscopy
The FTIR spectroscopy of P. aeruginosa AgNPs was performed in order to check the influence of bio-molecules which were responsible for the reduction, stabilization and capping of AgNPs as well as to determine the functional groups of the AgNPs. The completely dried samples of AgNPs were used in order to perform FTIR. The spectrum was recorded on FTIR IR Prestige-21 P/N 206-72010. SHI-MADZU in the transmission range of 4000-500 cm 1 24 .

Scanning Electron Microscopy SEM
The size and morphology of the P. aeruginosa AgNPs was analyzed by coating the air dried AgNPs and observing under scanning electron microscope EM6200 . 2.7.4 X-ray diffraction XRD Formation of P. aeruginosa AgNPs was further checked by XRD technique using an X-ray diffractometer Phillips PW 1729/40 operated at 40 kV, 40 mA, step size of 0.2, over the 2θ range of 20-80 . Glass slides coated with AgNPs were tested following manufacturer guidelines.

Statistical analysis
All experiments were performed in triplicates. Microsoft Excel 2019 was used to draw graphs while SPSS version 10 was used to calculate means, standard error and one way ANOVA test followed by Tureky s test was used to determine the significance level at p ≤ 0.05 23 . The figure of FTIR data was made using origin 2019A 8.5.1 . The pictures of SEM and XRD were derived from one replicate.

Synthesis of AgNPs
Biogenic synthesis of AgNPs by using four bacterial isolates E. coli, P. aeruginosa, B. subtilis and B. licheniformis was confirmed by the change of color from pale yellow to brown after 72 hours incubation compared to the two controls. The formation of AgNPs indicated that certain reducing agent released by the tested bacteria are actually involved in the reduction of Ag ions to AgNPs. In control group, the reduction of Ag ions did not occur due to the absence of reducing agent produced by bacteria, hence no color change was observed Fig. S1 .

Antibacterial activity of AgNPs
Biogenically synthesized AgNPs showed significant antibacterial activity against human pathogenic strains such as E. coli, P. aeruginosa, B. subtilis and B. Fig. 5 and Table 1 . The one-way ANOVA showed that P. aeruginosa AgNPs showed significant antibacterial activity p 0.05 as compared other biogenically synthesized AgNPs and positive control.

MIC and MBC determination
MIC was measured to determine the lowest concentration which is effective to inhibit bacterial growth. It was observed that MIC values of E. coli AgNPs ranged from 6.0 to 8.6 µg/mL, P. aeruginosa AgNPs from 2.6 to 3.3 µg/mL,

Characterization study 3.4.1 UV-visible spectroscopy
The P. aeruginosa AgNPs were analysed using UV visible spectrophotometer. The absorption spectrum for AgNPs was measured from 370 nm-770 nm which showed peaks from 400 nm-430 nm. The absorption peak was observed around 430 nm Fig. 1 . The P. aeruginosa AgNPs showed broad peaks due to variation in the size of the NPs. Increased peak intensity is the clear indication of increased NPs in the solution.

FTIR spectroscopy
The FTIR result of P. aeruginosa AgNPs showed number of bands in the region 4000-500 cm 1 . The spectrum analysis of AgNPs showed absorption bands at different peaks

Scanning Electron Microscopy SEM
The size and morphology of P. aeruginosa AgNPs was examined by SEM analysis. The P. aeruginosa AgNPs were mostly spherical in shape with size ranging from 32-46 nm scale bar of 0.5 µm . There were few NPs aggregation suggesting that the protein molecules may play crucial role as capping agent for NPs thus preventing agglomeration and providing stability to the synthesized NPs

Discussion
Biologically synthesized AgNPs have been proved as therapeutically effective and valuable compounds. They also have excellent antimicrobial and antiviral activity 1,25,26 . Although, there are a lot of techniques available which can be used for the synthesis of AgNPs. Much of these techniques were focused on use of chemicals. However, green synthesis or biogenic synthesis of AgNPs is more ecofriendly approach 26,27 . In the present study, four bacterial strains i.e., E. coli, P. aeruginosa, B. subtilis and B. licheniformis were used for the biogenic synthesis of AgNPs. Synthesis of AgNPs was confirmed by color change from pale yellow to brown. Previously, Gurunathan et al. 16 described color change to brown as confirmed biogenic synthesis of AgNPs. Likewise, Masum et al. 28 reported that the formation of AgNPs get confirmed by brown coloration and more saturated brown color indicates the more AgNPs in the solution. The change in color is due to the excitation of the surface plasmon vibrations in metal NPs 11 . After visual observation the solution was further analyzed by UV visible spectrophotometer.
Following formation, all four biogenically synthesized AgNPs were tested for antibacterial potential against four human isolates using agar well diffusion assay. It was observed that E. coli AgNPs showed ZOI from 16.0-19.6 mm against four isolates. P. aeruginosa AgNPs showed 19.0 to 22.5 mm ZOI, B. subtilis AgNPs showed 7.0 to 17.3 mm ZOI and B. licheniformis AgNPs showed 17.0 to 20.0 mm ZOI against all tested isolates. Among all biogenic AgNPs, P. aeruginosa AgNPs showed significant antibacterial potential even higher than positive control rifampicin 50 µg/ mL , because the size of P. aeruginosa synthesized AgNPs were 10-100 nm and showed strong antimicrobial effect against both Gram-positive and negative bacteria. The small particle size enables AgNPs to adhere to the cell wall and penetrate into the bacteria cell easily, which in turn improves their antimicrobial activity against bacteria Table  1 . The findings of this study corroborate with Aziz et al. 25 who observed the significant antibacterial potential AgNPs   29 reported that AgNPs release Ag ions from the surface and these ions are responsible for the bactericidal efficacy of AgNPs. The more Ag ions release will kill more bacteria which results in bigger ZOI. The observed ZOI by biogenically synthesized NPs were obtained after subtracting the ZOI by AgNO 3 , hence indicating the actual antibacterial potential of biogenic NPs. Silver nitrate has shown different effects against bacteria at high concentrations, killing bacteria by different mechanisms, which are binding to the thiol groups of protein and denaturing them, programmed cell death apoptosis and causing the DNA to be in the condensed form, which inhibits cell replication. While low concentrations 5-10 mM of AgNO 3 leads to the formation of silver NPs 30 . This study used low concentration 10 mM AgNO 3 for forming AgNPs. However, it s already stated that in depth antibacterial mechanism of NPs is still unclear. In particular, various bacterial isolates, their mechanism of action, resistance profile, and NP potential have been reported previously, which make it difficult to compare bacteriostatic vs bactericidal activity. In addition, no single method/criteria justify the exact through information about antibacterial mechanisms of tested NPs 31 .
In this study, tube dilution method was used for the determination of MIC and MBC. The maximum MIC value 8.6 µg/mL was noted against E. coli by E. coli AgNPs while lowest value was 4.3 µg/mL by P. aeruginosa AgNPs against B. licheniformis. Overall, P. aeruginosa AgNPs showed the lowest MIC and MBC values against all tested strains, hence subjected to characterization study. The MIC values were 4.3 to 8.6 µg/mL against E. coli, 6 to 7.6 µg/mL against P. aeruginosa, 4.3 to 6 µg/mL against B. subtilis and 4.6 to 6.6 µg/mL against B. licheniformis. These results were significant p 0.05 . The above results showed that AgNPs have lowest minimum inhibitory concentration against pathogenic bacteria suggesting the broad spectrum nature of their antimicrobial activity 32 . The MBC values were 5.6 to 11.6 µg/mL against E. coli, 6.3 to 8.8 µg/mL against P. aeruginosa, 5.6 to 10.3 µg/mL against B. subtilis and the MIC value against B. licheniformis was 5.5 to 8.3 µg/mL Table 2 . The findings of this study are consistent with the results by 33 .
The P. aeruginosa synthesized AgNPs show strong peek at 430 nm 28 . The occurrence of peek around 430 nm indicates the presence of AgNPs. Aziz et al. 25 reported that the reduction of silver nitrate causes the occurrence of peek around 430. Biosynthetic mechanism of AgNPs has been assumed that the Ag ions required NADPH-dependent nitrate reductase enzymes for their reduction, which were released by tested bacteria in their extracellular environment 34 .
FTIR of P. aeruginosa AgNPs was performed to check the involvement of biological molecules. The bands present at 3261, 3061, 2947, 2358, 1338 and 1394 cm 1 corresponded to the stretching vibrations of alcohol O-H , primary amines N-H , alkane C-H , amine C-N and alcohol C-O groups respectively, as reported previously by Liaqat 35 and Kalyanasundaram et al. 36 . The SEM data showed that silver nanoparticles are in spherical shape with average size of 40 nm. The formation of various sharp peeks in XRD, indicated the presence of different molecules, involved in the stabilization of the NPs. Similar result was reported by Kumar et al. 37 .

Conclusion
This study showed the effective antibacterial potential of biogenically synthesized AgNPs and suggested these as alternative source of antimicrobial agents against human pathogenic bacteria. AgNPs showed strong bactericidal effect even at lower concentration against the test human pathogenic strains. Ongoing in vivo study will prove their toxicity analysis and safety in pharmaceutical and therapeutic biomedical applications.

Conflict of Interests
On behalf of all authors, the corresponding author states that there is no conflict of interest.

Author Contributions
I. Liaqat designed the project and supervised the complete study. S. Tufail did experimental work on characterization and wrote the first draft. I. Liaqat and S. Tufail did the statistical analysis. I. Liaqat wrote the final draft. S. Andleeb, A. Bibi, I. Liaqat, and S. Naseem proof read the manuscript. S. Ibrahim, M. Tahir, G. Saleem, and A. Haleem helped in experimental work. All authors approved the final version of manuscript.

Supporting Information
This material is available free of charge via the Internet at doi: 10.5650/jos.ess21291