Comparative Account of Biogenic Synthesis of Silver Nanoparticles Using Probiotics and Their Antimicrobial Activity Against Challenging Pathogens

The present work focusses on development of a safe, inexpensive, and more accessible source for biosynthesis of silver nanoparticles. Four different in-house probiotic isolates, i.e., Lactobacillus pentosus S6, Lactobacillus plantarum F22, Lactobacillus crustorum F11, and Lactobacillus paraplantarum KM1 isolated from different food sources, were used in the current study to check their ability to synthesize silver nanoparticles. All the probiotic-synthesized silver nanoparticles show maximum surface plasmon resonance (SPR) at a peak of 450 nm, which confirms the formation of silver nanoparticles. Scanning electron microscopy (SEM) analysis identified the shape and distribution of silver nanoparticles. Transmission electron microscopy (TEM) revealed the average size of synthesized nanoparticles in the range of 10–50 nm, with the smallest size of 5 nm for silver nanoparticles synthesized by L. crustorum F11. Further, Fourier-transform infrared spectroscopy (FTIR) detected the presence of different functional groups responsible for reduction of silver ion to form silver nanoparticles. The antimicrobial activity of these AgNPs was also found to be effective against different bacterial and fungal pathogens, viz., antibiotic-resistant Staphylococcus aureus, Bacillus cereus, Listeria monocytogenes, Pythium aphanidermatum, Fusarium oxysporum, and Phytopthora parasitica. However, L. crustorum F11–synthesized AgNP showed maximum inhibition against all the bacterial and fungal pathogens, with highest against S. aureus (20 ± 0.61 mm) and F. oxysporum (23 ± 0.37). Findings from this study provide a durable and eco-friendly method for the biosynthesis of silver nanoparticles, having strong antimicrobial activity against different multidrug-resistant microorganisms.


Introduction
The budding resistance in the harmful pathogenic microorganisms against the current antibiotics and drugs is an alarming concern. We see a huge spike in multidrug-resistant organism (MDRO) population which has rendered myriad of infections treatments futile. MDROs are multiplying at faster rate, making various health-related infections worse and almost untreatable [1][2][3]. Nanotechnology is one field of science that has put its foot forward for the rescue. Nanotechnology is a renowned field of science that has witnessed exponential growth and evolution, in general and industrial application since the last century. Apparently, nanoparticles have shown potential in averting and/or subduing the infections caused by these pathogenic microorganisms. There are different physical and chemical methods employed already for the synthesis of nanoparticles, related to hazardous chemicals exhibiting toxicity [4]. Biologically synthesized nanoparticles have an upper hand over these conventional methods as they are eco-friendly, cost-effective, and less hazardous. Among different metallic nanoparticles, silver nanoparticles (AgNPs) are gaining attention as one of the most promising nanoparticles due to various unique properties it possesses [5]. AgNPs have large surface area to volume ratio and have strong toxic effect against wide range of microorganisms and can be used as a potential antimicrobial agent [6]. Silver nanoparticles synthesized using lactic acid bacteria (LAB), have been reported in different studies nowadays [7]. Probiotics are live microbes which, when administered adequately, can lead to healthier benefits to the host cells [8,9]. Lactobacillus and Bifidobacterium are the most used probiotic strains to synthesize silver nanoparticles. Probiotic synthesized silver nanoparticles have been found to possess effective antimicrobial efficacy against a wide range of pathogenic microbes and have enhanced surface area to volume ratio, high catalytic capabilities, and tendency to generate reactive oxygen species [10].
As the use of probiotics in synthesizing silver nanoparticles is a novel and current trending research, therefore, in the present paper, an attempt has been made to biosynthesize silver nanoparticles from four in-house potential probiotic cultures, i.e., Lactobacillus pentosus S6, Lactobacillus plantarum F22, Lactobacillus crustorum F11, and Lactobacillus paraplantarum KM1 that have already been isolated from different food sources and their characterization by using UV-Visible spectroscopy, scanning electron microscopy (SEM), transmission electron microscopy (TEM), and Fourier-transform infrared spectroscopy (FTIR). Further their antagonistic spectrum was examined against different challenging pathogens (bacteria and fungi).

Materials and Methods
The four in-house probiotic cultures isolated from different food sources were collected from Microbiology Laboratory, Department of Basic Sciences, UHF Nauni, Solan, H.P, India, for the biosynthesis of silver nanoparticles and analysis of their antimicrobial potential. The bacterial strains used for antimicrobial analysis in the study were obtained from MTCC (IMTECH, Chandigarh), CRI (Kasauli), and IGMC (Shimla). All the chemicals used in this study were of analytical grade.

Biosynthesis of Silver Nanoparticles Using Probiotic Isolates
The collected probiotic strains were inoculated in 100 ml of MRS media and incubated overnight at 37 °C for 24 h. After incubation, the culture was centrifuged at 10,000 rpm for 15 min. Ten milliliters of culture supernatant from each isolate was separately mixed with 90 silver nitrate (AgNO 3 ) solution (0.1 mM) and another reaction mixture with only silver nitrate was used as control. The prepared solutions were incubated at 30 °C (24 h) and kept in dark to avoid any photochemical reactions during the investigation. The change in color in synthesized silver nanoparticles was observed after 24 h. The biosynthesized AgNPs were then characterized by using UV-Vis, SEM, TEM, and FTIR spectroscopy.

UV-Visible Spectroscopy Analysis
The bioreduction of silver ion in aqueous solution was monitored by periodic diluted sampling of aliquots and subsequently measuring the spectra of the solution on UV-Vis spectrophotometer [11]. The absorbance of all the four synthesized AgNP was read at different wavelengths, i.e., 350, 450, 550, and 650 nm.

Scanning Electron Microscopy (SEM)
Thin film of silver nanoparticles was prepared on aluminum plate by dropping a small amount of the sample on the plate; extra solution was removed using blotting paper. The film on aluminum plate was dried overnight. SEM analysis was performed on a JEOL; model JSM -6610LV instrument operated at an accelerating voltage of 20 keV [11].

Transmission Electron Microscopy (TEM)
Transmission electron microscopy was performed by using model FP 5022/22-Tecnai G2 20 S-TWIN. TEM analysis was performed by placing a drop of the synthesized AgNP suspension on a carbon-coated copper grid and allowing the water to evaporate inside a vacuum dryer for 1 h.

Fourier-Transform Infrared Spectroscopy (FTIR)
For FTIR analysis, small amount of synthesized AgNP was dried in a freeze dryer (lyophilizer) for 24 h; then, freezedried sample was appointed with KBr pellets and analyzed using a Thermo Nicolet model: nexus 870 in range of 450-4000 cm −1 at a resolution of 4 cm −1 [12].

Antibacterial Activity of Silver Nanoparticles
Antibacterial assay of synthesized silver nanoparticles was performed against three standard test indicators, i.e., antibiotic-resistant Staphylococcus aureus IGMC, Listeria monocytogenes MTCC839, and Bacillus cereus CRI using well plate assay [13]. The inoculum of pathogen was streaked on a nutrient agar plate using sterilized swabs. Wells were punched on the plate with the help of a sterile borer and 100 µl of biosynthesized silver nanoparticles (concentration: 200 µg/ml) was added to each well. The plates were then incubated at 37 °C for 24 h; the experiment was performed in triplicate.

Antifungal Activity of Silver Nanoparticles
Antifungal activity was checked against three fungal pathogens, viz., Pythium aphanidermatum, Fusarium oxysporum, and Phytopthora parasitica by well plate assay using dual culture technique [14]. Seven-day-old culture bit of indicator fungi was placed on one side of potato dextrose agar (PDA) plate and 100 µl of 0.1 mM AgNP was added in the well on the other side of the plate. The plates were incubated at 28 ± 2 °C for 7 days and observed for inhibition of mycelial growth produced around the well. The experiment was performed in triplicate.

Statistical Analysis
Analysis of variance (ANOVA) was performed, and values were represented as a mean of three replicates (n = 3) ± SD. The significance level was maintained as P-value < 0.05.

Biosynthesis of Silver Nanoparticles
Silver nitrate was used as a precursor for the biosynthesis of silver nanoparticles. All the four probiotic isolates were capable of synthesizing AgNPs as all of them showed color change, i.e., reduction of Ag + to AgNPs after incubation as compared to the control sample (AgNO 3 ) (Fig. 1a). This color change was considered the first distinctive feature of the formation of AgNPs and is due to excitation of surface plasmon resonance (SPR) in the AgNPs [15]. Many researchers nowadays are exploring the role of probiotic microorganisms for the synthesis of biocompatible AgNPs and determining their applications in health sector. Recently, Ysouf et al. [5] reported the utilization of biomass from Lactobacillus plantarum TA4 for the biosynthesis of AgNP. Different Probiotic bacteria like Bifidobacterium animalis, Lactobacillus acidophilus, and Streptococcus thermophilus isolated from milk products were also used to synthesize silver nanoparticles [16]. Furthermore, for the confirmation of synthesis of AgNPs, UV-Visible spectroscopy analysis was used. The absorption spectrum was recorded in different wavelength ranges, i.e., 350, 450, 550, and 650 nm, respectively (Fig. 1b). The highest absorbance peak for all the four isolates was found at the wavelength of 450 nm, which was in the range specific for AgNP synthesis. Malathi et al. [17] monitored the synthesis of AgNP from Lactobacillus sp. by UV-spectroscopic analysis and observed peak between 400 and 450 nm indicating the presence of silver nanoparticles.

SEM and TEM Analysis
SEM reveals the external morphology and texture, whereas TEM is a valuable tool used to visualize size of synthesized AgNPs. Figure 2a, b, c, and d shows the SEM analysis images of AgNPs synthesized from L. pentosus S6, L. plantarum F22, L. crustorum F11, and L. paraplantarum KM1 respectively. AgNPs were found to be spherical in shape and were present as an individual particle and/or in aggregates. Production of silver nanoparticles from Lactobacillus sp. with varying shapes, i.e., rectangular to spherical, was also cited in the literature [18]. Figure 3a, b, c, and d and Table 1 show the TEM image analysis of synthesized AgNPs. Silver nanoparticles of different size range were observed in our study. Both the L. pentosus S6 and L. paraplantarum KM1-synthesized AgNPs had a mean size of 50 nm, whereas 20 nm for L. plantarum F22 AgNP and 10 nm for L. crustorum F11 AgNP respectively. In several studies where lactic acid bacteria and other microorganisms were used to synthesize AgNP, the particle size was found to be in 20-100-nm range [19,20].

Fourier-Transform Infrared Spectroscopy (FTIR) Analysis
FTIR spectrum is used to identify different functional groups, responsible for the reduction and stabilization of silver ions to silver nanoparticles. FTIR spectrum of synthesized AgNPs was presented in Fig. 4a, b, c, and d respectively. L pentosus S6-synthesized AgNPs (Fig. 4a) show absorbance peak at 1710 cm −1 which corresponds to C = O stretching vibration of ketone. The absorbance peak at 2273 cm −1 corresponds to N = C = O stretching and presence of isocyanate functional group, whereas absorbance peak at 1367 cm −1 indicated C-O stretching vibration of an ester [21]. L. crustorum F11 synthesized silver nanoparticles (Fig. 4b) show absorbance peak at 3222 cm −1 referred to O-H stretching and presence of carboxylic acid, whereas absorbance peak at 1628 cm −1 revealed C = C could be due to amide II vibrations [22]. The peak at 1146 cm −1 represents O-H bond of the aliphatic ether, 1062 cm −1 corresponds to O-H stretch of primary alcohol, and 1925 cm −1 peak indicated C = C = C stretch of allene. For silver nanoparticles synthesized  from L. plantarum F22 (Fig. 4c), the absorbance peak at 1695 cm −1 corresponds to C = N stretching of imine group. The absorbance peak at 1324 cm −1 could be attributed to C-N bond of aromatic amide, and absorbance peak at 1635 cm −1 represented C = C stretching of alkenes. Silver nanoparticles synthesized by L. paraplantarum KM1 (Fig. 4d) show peak at 1695 cm −1 had C = O stretch of conjugated aldehyde and peak at 1941 cm −1 was identified as C-H stretch of aromatic compound. The peak at 2161 cm −1 represented S-C = N stretch of thiocyanate and 1647 cm −1 peak pointed out the C = N stretch of sulfone. All the four probiotic isolates used in the study contain important functional groups (hydroxyl, protein, and carboxyl) in the cell membrane, which plays an important role in the reduction of silver nitrate and thus capable of biosynthesizing silver nanoparticles.

Antibacterial Efficacy of Silver Nanoparticles
Strong antimicrobial activity was exhibited by different probiotic-synthesized AgNPs against the three pathogens, i.e., Staphylococcus aureus, Bacillus cereus, and Listeria monocytogenes. Among all, however, L. crustorum F11-synthesized AgNPs showed maximum zone of inhibition against all the microbial strains with maximum zone of inhibition of 20 ± 0.61 mm against S. aureus, 14 ± 1.01 mm for L. monocytogenes, and 12 ± 7.07 mm for B. cereus respectively (Fig. 5). Kumar

Antifungal Activity of Probiotics Synthesized Silver Nanoparticles
The antifungal activity was tested against three fungal pathogens, viz., Pythium aphanidermatum, Fusarium oxysporum, and Phytopthora parasitica. Here also, L. crustorum F11-synthesized AgNP showed maximum inhibition against all the three fungal pathogens, i.e., 23 ± 0.37 for F. oxysporum, 20 ± 1.01 mm against P. parasitica, and 32.6 ± 0.48 mm against P. aphanidermatum F11 (Fig. 6). Matei    antimicrobial activity; however, the one synthesized from L. crustorum F11 stands apart with maximum antagonistic spectrum against different bacterial and fungal pathogens. This strong inhibitory effect could be attributed to its smaller size (10 nm) in comparison to the other probiotic-synthesized AgNPs. Two similar studies conducted by Lu et al. [25] and Morones et al. [26] have revealed a direct relation between antimicrobial activity and size of the AgNPs. They both concluded that AgNPs have stronger antagonistic activity for the sizes ranging in 1 ~ 10 nm as compared to their counterparts in larger sizes. Small particle sizes help AgNPs adhere to the cell wall and for easy cell penetration, increasing antimicrobial activity against harmful pathogenic microorganisms. AgNPs synthesized in this research are easy to use and ecofriendly and exhibit antimicrobial properties; it makes them suitable for health care industry. Much work needs to be done, however, to improve efficacy, maintain particle size, and alleviate safety concerns.

Conclusions
This study exhibited silver nanoparticle biosynthesis using different health beneficial probiotic isolates. Initial confirmation of AgNP synthesis was confirmed by color change and UV-Visible spectroscopy analysis. Furthermore, SEM and TEM analysis depicted the morphology and size (20, 10, and 50 nm), and FTIR analysis identified different organic compound responsible for reduction and formation of AgNPs. The synthesized AgNPs were found to be safe and economical and showed strong antimicrobial potential against challenging microbial strains. In conclusion, our study recommends using probiotics for AgNP formation having promising results against health deteriorating multidrug resistant microorganisms.