Physiochemical properties and antibacterial activity of silver nanoparticles green synthesized by Camellia sinensis and Prunus africana extracts

Antibiotics have been the nucleus of chemotherapy since their discovery and introduction into the healthcare system in the 1940s. They are used routinely not only to treat bacterial infections but also to prevent infections in patients with compromised immune systems and enhancing growth in livestock. However, resistance to last-resort antibiotics used in the treatment of MDR infections has been reported worldwide. Therefore, the aim of this study was to evaluate green synthesized nanomaterials such as AgNPs as alternatives to antibiotics. UV Vis Spectroscopy surface plasmon resonance peaks for AgNPs were obtained between 417 to 475nm. XRD analysis generated 4 peaks for both PAE and CSE biosynthesized AgNPs positioned at 2θ angles of 38.2˚, 44.4˚, 64.5˚, and 77.4˚ corresponding to crystal planes (111), (200), (220) and (311) respectively. DLS registered mean zeta potential of + 6.3mV and + 0.9mV for PAE and CSE biosynthesized nanoparticles respectively. FTIR spectra exhibited bands corresponding to different organic functional groups con�rming capping of AgNPs by PAE and CSE phytochemicals. FESEM imaging showed that AgNPs were spherical with average size distribution ranging from 10 to 19nm. Biosynthesized AgNPs exhibited maximum growth inhibitory zones of 21mm with MIC and MBC of 125µg/ml and 250µg/ml respectively against carbapenem resistant bacteria.


Introduction
The greatest challenge of our generation and generations to come is antimicrobial resistance as different pathogenic bacteria have continuously evolved to become resistant to even the most recently synthesized antibiotics.This scenario has complicated treatment outcomes even to the commonest bacterial infections (Livermore et al., 2011).The U.S. Centers for Disease Control and Prevention (CDC) approximates that antibiotic resistance is accountable for more than 2 million infections and 23,000 deaths each year in the United States, at a direct cost of $20 billion and extra output losses of $35 billion (CDC, 2013).In Europe, an estimated 25,000 deaths are attributable to antibiotic resistant infections, costing €1.5 billion annually in direct and indirect costs (ECDC., 2017).Even though there is no trustworthy data on economic losses in the third world countries, Studies in India (Laxminarayan, Boeckel, & Teillant, 2015), Tanzania and Mozambique (Kayange, Kamugisha, Mwizamholya, Jeremiah, & Mshana, 2010;Roca et al., 2008) point out that increased deaths in neonates are attributed to antibiotics resistant infections.
Repeated attempts to develop alternative approaches such as use of herbal medicines; sh mucins and nanotechnology have been made.Of all, the most promising novel therapeutic option in this present scenario is the application of nano-scale materials as antimicrobial agents as they have exhibited very high surface area to volume ratio and the exceptional chemical complexities.
The nanotechnology eld is anticipated to be the gate way in combating both infectious and non-infectious diseases (Albrecht, Evans, & Raston, 2006;Morones et al., 2005).Among the metallic nanoparticles, Silver nanoparticles (Ag NPs) owing to their potent antimicrobial activity against multidrug resistant pathogens ranging from viruses, prokaryotes to eukaryotes, have become the center of attention of robust research (Gong et al., 2007).Silver NPs exhibited great antibacterial e cacy against Vibrio cholera, Salmonella typhi Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa in previous experiments (W.-R.Li et al., 2010;Morones et al., 2005).In the 1960s and 70s, a combination of silver nitrate and sulfonamide to form silver sulfadiazine cream with broad-spectrum antimicrobial activity was the rst option for topical treatment of burns as its e cacy against E. coli, S. aureus, Klebsiella sp., and Pseudomonas sp.fungal and viral infections was laudable (Fox & Modak, 1974;Moyer, BRENTANO, GRAVENS, MARGRAF, & MONAFO, 1965).However, Nanoscale Silver in comparison to non-nanoscale one such as silver nitrate, presents concurrently high solubility, great chemical reactivity, and formidable broad-spectrum bacteria growth inhibitory activity at very low concentration (Agnihotri, Mukherji, & Mukherji, 2013;Bondarenko et al., 2013;Martinez-Gutierrez et al., 2013;Panáček et al., 2006).Most importantly, the bactericidal action of nanoparticles is reliant on physicochemical properties such as size and shape hence variability in the mode of action of different forms of nanoparticles may enlighten why resistance to this treatment is yet to be reported (Kvítek et al., 2008;Marková et al., 2012).To date, the bactericidal effect of nanoparticles (NP) is yet to be fully clari ed.However, it might reside within the capacity of nanoparticles to discharge cations from nano-prearranged surfaces, which irreversibly disorganize bacterial cell wall, inactivate vital proteins, chelate DNA and lead to generation of reactive oxygen species known to have high microbicidal activity (N.Durán, Nakazato, & Seabra, 2016;Rizzello, Cingolani, & Pompa, 2013;Wang, Hu, & Shao, 2017;Yin et al., 2020).
Furthermore, the conventional approaches employed to fabricate nanoparticles such as chemical and physical methods have limitations.Physical approaches are not cost effective because they regularly require extremely expensive equipment, high temperature and pressure in addition to high energy consumption (Guzmán, Dille, & Godet, 2009) hence making them unpopular in third world countries.Chemo nanoparticle fabrication mainly entails the models of wet chemistry where several chemical reducing agents are exploited to reduce metal salts in solutions (Polavarapu & Liz-Marzán, 2013;Tahir et al., 2013).
The main drawbacks faced by chemical methods include use of costly metal salts in addition to hazardous solvents and reducing agents.Therefore, nanoparticles synthesized by chemical methods exhibit nonselective toxic to both target bacterial and host cells (Kawata, Osawa, & Okabe, 2009).
Additionally, numerous stabilizers are also necessary to thwart aggregation of nanoparticles to make them physiologically compatible and functional (Castro, Blázquez, González, & Ballester, 2014).
Due to challenges faced by conventional methods used in fabrication of nanoparticles, researchers have been motivated to invent novel, cost effective, uncomplicated eco-friendly biological approaches.Several biological approaches such as biosynthesis of nanoparticles using bacteria (Dickson, 1999), fungi (Nelson Durán, Marcato, Alves, De Souza, & Esposito, 2005) and plant extracts (Huang et al., 2007) have been reported.The most robust and popular of all is green synthesis of inorganic nanoparticles exploiting green plant extracts with antioxidant activity.The antioxidant properties of phenolic phytochemical are mainly attributed to their reducing abilities that permit them to operate as reducing agents and singlet oxygen scavengers, therefore reducing metal salts to nanoscale by scavenging electron from them.Presently possible sources of phytochemicals have been broadly studied in various plant species and plant parts including leaves, vegetables, fruits, oil seeds, herbs, barks, and roots, as well as in the extracts of entire plants (S.Li et al., 2007).Moreover, phytochemicals effectively support the green generation of nanoparticles by acting as reductants as well as functionalizing the resultant nanoparticles (Khan et al., 2013).Furthermore, the green based synthesis and functionalization of nanoparticles can be easily executed under normal physiological condition (Huang et al., 2014).Without a doubt, due to the double effect of phytomolecules, green based synthesis meets the criteria of the best approach for preparing nanoparticles for biological applications because the resultant nanoparticles can be instantly applied without any post synthesis modi cation (Mie et al., 2014).This study examined the e cacy of Camellia sinensis and Prunus africana bark extract mediated green synthesized AgNPs against carbapenem resistant (CR) E. coli and K. pneumoniae.

Site description and source of materials
This study was carried out at the Pharmacology and Microbiology Laboratories, College of Veterinary Medicine, Animal Resources and Biosecurity, Makerere University, Materials Research Department (MRD), iThemba LABs, Cape Town and Nanotechnology Unit, department of Chemical Engineering, University of South Africa, Florida Campus.Processing of plants material, phytochemical extraction and green synthesis of Ag NPs were conducted from the Pharmacology laboratory and MRD while Ag NPs susceptibility assays were executed from the Microbiology Laboratory.Characterization of Nanoparticles was accomplished from MRD and Nanotechnology Unit.Con rmation of the identity of Prunas africana was done from department of Botany, Makerere University.Silver nitrate (AgNO 3 ) was obtained from Sigma Aldrich, USA, Camelia sinensis leaves were purchased from Igara Tea Estates, Bushenyi while Prunas africana bark and leaves were acquired from Maramagambo forest which covers the southern part of Queen Elizabeth National park located in Bushenyi district, Uganda.

Extraction of phytochemicals from Camellia sinensis and Prunus africana
Leaves of Prunus africana were analyzed by a botanist at department of Botany Makerere University to con rm its identity.To obtain Camelia sinensis extract (CSE) and Prunas africana extract (PAE), plant materials (leaves for CSE and the bark for PAE) were washed and lichens scrapped off, dried under shade on clean drying tables.The plant materials were then shredded into smaller particles 1-2cm 2 using knife and Pulverized into powdered form using electric laboratory grinder.Four hundred grams (400 g) of each powdered plant material were dissolved in 1.5L of sterile distilled de-ionized water in two different extraction bottles and left to stand for 5 days under darkness with daily occasional agitation for homogeneous mixing and extraction.The crude extract was sieved, and the ltrate concentrated by evaporation over steel pans in an oven at 37°C.After evaporation, the dry solid concentrate was scrapped off the pans, then transferred into clean top screw 50ml falcon tubes labeled as CSE and PAE and then stored 4°C.

Synthesis of silver nanoparticles using CSE and PAE
The Ag NPs were photosynthesized by adding 1ml (2%) of CSE concentrate into a round bottom ask containing 49 ml aqueous solution comprising 85mg of silver nitrate (0.5mM).The experiment was replicated to assess the effect of the concentration of plant extract on the yield and characteristics of nanoparticles by using different concentrations (4%, 8% and 16%) CSE.The above procedure was repeated using PAE, Table 1.UV Vis absorbance was taken immediately after mixing the plant extract and silver nitrate solution.For further absorbance reading, 5ml from each sample was dispensed into 15ml falcon tubes and kept in a dark cupboard.The round bottomed asks were placed in a dark cabinet and left to stand for 24 hours, after which each was equipped with a magnetic stir bar and xed with a cooling condenser.The reaction mixture was left to stand for 2 hours at 85°C, then left to cool down at room temperature followed by centrifuging at 9000 rpm for 30 minutes.The sediment obtained was washed numerous times with distilled de-ionized water after which the nal precipitate produced was dried at 80°C in an oven for 12 hours.

X-Ray diffraction Analysis (XRD)
About 500mg of green synthesized Ag NPs powder were analyzed with powder X-ray diffraction (XRD) employing BRUKER AXS diffractometer, D8 Advance (Germany) tted with Cu-Ka radiation (λKα 1 =1.5406Å) from 2θ = 0.5° to 130°, with increment D2J: (0.034°), voltage of 40 kV, current of 40 mA, power of 1.6 kW and counting time of 0.5 sec/step.Generated data was analyzed by OriginPro and resultant peaks 2 theta values were compared with the standard Ag NPs values from the International Center for Diffraction Data (ICDD) database.The average crystal particle size was calculated using Debye-Scherrer's formula given as; Where ø is the crystal particle size, β is full width at half maximum (FWHM), λ is the X-ray wavelength, is angle subtended in peak Fourier transform infrared spectroscopy (FTIR) Fourier transform Infrared spectroscopy with a PerkinElmer Spectrum RX I Fourier transform IR system with a frequency ranging from 400 to 4000 cm -1 and a resolution of 4cm -1 , set to perform at least 64 scans per sample was used to investigate the organic functional groups of the plant extracts used in the bioreduction of silver nitrate to silver nanoparticles as follows; 2 mg of silver nanoparticles and 2 g of Potassium bromide (KBr) were desiccated at 200°C under reduced pressure over night.The dried silver nanoparticles were standardized with 100 mg of KBr and then hard pressed to form very thin transparent circular pellets.The pallets were screened at 4000-400 cm -1 Wavenumber range.A KBr pellet was used as to plot the baseline.

Dynamic light scattering (DLS) and zeta potential
Zeta sizer Nano ZS Malvern Panalytical was used to evaluate the size distribution and zeta potential of silver nanoparticles.The Zeta sizer Nano ZS instrument was set to perform 60 scan times three times per sample to obtain mean size distribution of nanoparticles.The DLS technique was employed to analyze the nanoparticle size distribution according to standard method with some modi cations (Chattopadhyay et al., 2013a).The concentration of silver nanoparticles of 100 µg/mL was sonicated for 5 minutes to disaggregate the nanoparticles, and dynamic particle sizes were determined by suspending 0.5 µl of the sonicated nanoparticle suspension in 1ml of Millipore water in zetasizer cuvette followed by scanning of the nanoparticle suspension by a DLS analyzer.The zeta potential of silver nanoparticles was measured by Zetasizer-Nano ZS using the electrophoretic light scattering technology where 1 mg/mL of nanoparticle suspension was prepared in Milli-Q water in a 900 µl zeta sizer disposable cell.The suspension was screened 60 times per scan for three scans to compute the mean zeta potential of silver nanoparticles.

Scanning electron microscopy (SEM)
The size and morphology of the biosynthesized AgNPs were examined by Field Emission Scanning Electron Microscopy (FESEM), Carl Zeiss SIGMA model operated at 5 kV.Brie y, a thin lm of AgNPs was prepared by spreading 1 mg of each sample on carbon tape followed by coating with carbon.Surface images were captured at different magni cations.ImageJ software was used to estimate the size distribution of AgNPs.
Silver Nanoparticles susceptibility bioassay: Antibacterial activity was assessed by agar well diffusion method.
Carbapenem resistant E. coli ATCC 96522 and K. pneumoniae NTCT 9633 obtained from the archives of Microbiology department CHS, Makerere University were resuscitated in Tryptone soy broth (TSB) and incubated anaerobically at 37°C for 24 hours, then sub cultured on MacConkey agar plates anaerobically at 37°C for overnight.The fresh all night grown cultures were used to prepare a McFarland's turbidity standard equivalent to 0.5 McFarland's units.E. coli and K. pneumoniae lawns were inoculated on two separate sterile Muller Hinton Agar plates with a sterile spreader according to manufacturer Oxoid UK using the adjusted McFarland's turbidity standard.Three wells labeled positive control (+ve), negative control (-ve), and Ag NP were bore into the agar plates containing the bacterial lawns using sterile cork borer.Using a dilution factor of 1:2, Ag NPs were diluted using DMSO in bejou bottles and mixed thoroughly and 50µl of the resultant diluent was pipetted into the well labelled Ag NPs.Iminepem disk was used as a positive control while distilled water in DMSO was used as a negative control.The setup was left to set for a minimum of 30 minutes and then incubated at 37°C for 24 hours anaerobically and then checked for antibacterial activity.The zone of clearance for positive control (+ve), negative control (-ve), AgNPs were measured and recorded in millimeters.The experiment was replicated thrice to get the average inhibitory zone.

Determination of Minimum Inhibitory Concentration (MIC) of photosynthesized AgNPs
To assess the MIC, 0.1 g of AgNPs was dissolved in 0.6ml of sterile distilled de-ionized water in a sterile Eppendorf tube and mixed thoroughly by vortexing to form a solution.Then a setup of 8 wells of each extract on the micro titer plate was lled with 0.05ml of sterile TSB.Two-fold serial dilution was carried out on by transfer of 0.05ml of the Ag NP solution from the rst well to the eighth well on the micro titer plate containing 0.05ml of sterile Tryptic soy Broth (prepared according to manufacturer Oxoid UK).0.5µl of 0.5McFarland's turbidity standard prepared by suspension of fresh bacterial culture of E. coli and K. pneumoniae in separate test tubes, were dispensed into their corresponding wells containing diluted Ag NPs and incubated aerobically at 37°C for an overnight followed by plating of samples from each well on MHA and left to all night at 37 o C under aerobic conditions.MIC was determined from the dilution factor with minimum growth inhibition.

Determination of Minimum Bactericidal Concentration (MBC) of biosynthesized AgNPS
Minimum bactericidal concentration was determined by further performing a tenfold serial dilution (1/10 and 1/100) for the dilutions that didn't show growth under MIC determinations on the MHA plate.The two dilutions were further plated on MHA plates that were incubated at 37°C.After incubation the plates were observed for growth, MBC was determined from the dilution next to one which has growth.

Data analysis
Data analysis was done using Origin version 2019b.Comparison of mean zone (diameter in millimeter) of inhibition for CSE-AgNPs and PAE-AgNPs was computed by one-way ANOVA.A P-value of ≤ 0.05 indicated substantial statistical variance.
Increase in the plant extracts concentration resulted into gradual broadening and shifting of the SPR peaks towards the long wavelength; there was a shift of SPR peaks from 434 nm for 2% PAE to 472 nm for 8% PAE.Likewise, a shift from 430 nm for 2% to 461 nm for 8% and 16% CSE was registered, but more prominent in PAE photosynthesized nanoparticles.Identical UV Vis absorbance spectra were observed for 8% and 16% CSE synthesized nanoparticles, Tables S1 and S2; Figs 1-3.Furthermore, it's worth noting that the SPR for AgNPs was attained after 30 minutes with a high absorbance of 3.10 arb.units for CSE and at 4 hours, 6 hours and 8 hours with very low absorbance for 4%, 8%, and 2% ;16% PAE respectively.However, 16% PAE had the highest absorbance of 2.40 at 12 hours, Tables S1 and S2.Furthermore, SPR bands between 260 nm and 300 nm where registered, Figs 2 and 3 111), ( 200), ( 220) and ( 311) respectively.Peak intensity ranged from 1004 to 2824 arb.units and 313 to1174 arb.units for CSE and PAE mediated synthesized Ag NPs.From XRD patterns, the average crystallite size of the silver nanoparticles was computed using the Debye-Scherrer formula.The average crystallite size of PAE and CSE green synthesized silver nanoparticles ranged from 9 to 32 nm (mean = 17 nm) and 13 to 29 nm (mean = 21 nm) respectively, Table 2; Fig 5. Fourier Transform Infra-red Spectrometry Analysis FTIR spectra of PAE and CSE exhibited similar bands within wavenumber ranges (cm -1 ) of 3650-3400, 2960-2850, 2349, 2140-1990, 1650-1550, 1370-1390 and 800-400.Furthermore, Ag NPs biosynthesized by CSE were also capped by unique functional groups with spectra wavenumber ranges (cm -1 ) of 1390-1370 and 1250-1020 while PAE green synthesized Ag NPs exhibited distinctive organic functional groups within wavenumber series (cm -1 ) of 4000-3700, 1560-1500, 1440-1395, 1310-1250 and 1124-1087, Table 3; Fig 6.Dynamic light scattering revealed that the size distribution of silver nanoparticles biosynthesized by CSE ranged from 1 nm to 191 nm with mean diameter of 51 nm and mean zeta potential of +0.9 mV.For PAE green synthesized nanoparticles, size distribution ranged from 1nm to 220 nm with average diameter of 59 nm and mean zeta potential of +6.3 mV

Surface morphology analysis
Surface morphology analysis by FESEM shows that PAE and CSE biosynthesized AgNPs were mainly spherical in shape and aggregated in layers, Fig, 7. As the concentration of the plant extracts used increased from 2% to 16%, the level of aggregation increased to form large clusters with slimy material on the surface and between individual nanoparticles and aggregates and is more evident for PAE photosynthesized nanoparticles.Nanoparticle size computation by ImagaJ software from FESEM images showed that the nanoparticles size distribution ranged from approximately 3 nm to 98 nm and 4 nm to 94 nm for CSE and PAE biosynthesized AgNPs respectively.The average size distribution of nanoparticles decreased from 10 nm to 16 nm and 13 nm to 19 nm for CSE-AgNPs and PAE-AgNPs respectively as the concentration of plant extracts increased from 2% to 16%.
AgNPs susceptibility assay PAE and CSE green synthesized AgNPs demonstrated potent antibacterial activity with statistically similar growth suppression zones of up to 21 mm for carbapenem resistant (CR) and sensitive E. coli and K. pneumoniae.Furthermore, low MICs and MBCs were registered for the biosynthesized AgNPs, Tables 4-5.

Discussion
The reduction process of silver ions to Ag NPs was indicated by change in color of the reaction mixture from pale brown to dark brown for PAE and dark blue for CSE which was observed macroscopically.Additionally, Ag NP synthesis was con rmed and monitored with UV Vis Spectrophotometry.UV-Vis spectroscopy is a valuable tool for basic characterization and monitoring of the process of nanoparticle synthesis and stability.This is due to the distinctive optical properties of nanoparticles that enable them to intensely interact with speci c wavelengths of UV visible light spectrum (Kelly, Coronado, Zhao, & Schatz, 2003;Lee & El-Sayed, 2006;Sastry, Patil, & Sainkar, 1998).Metal surfaces are comparable to plasma, hence possess free electrons in the conductance region and positively charged nuclei.In Ag NPs, the forbidden gap between conduction and valence bands is very minute which allows free movement of electrons.This free movement of electrons generate a surface plasmon resonance (SPR) absorption band, owing to the mutual oscillation of Ag NPs electrons in resonance with the light wave (Das, Nath, Chakdar, Gope, & Bhattacharjee, 2010;Tomaszewska et al., 2013).In this study, the primary characterization and evaluation of CSE and PAE mediated Ag NPs green synthesis by UV Vis absorbance spectral was successful.UV Vis spectroscopy registered sharp absorbance SPR band at wavelengths ranging from 436 nm to 475nm for PAE and 430 nm to 456 nm for CSE which is a unique optical property for Ag NPs (Njagi et al., 2011).Comparable ndings were reported in other phytochemical mediated Ag NPs green synthesis studies (Anandalakshmi, Venugobal, & Ramasamy, 2016;Kagithoju, Godishala, & Nanna, 2015;Mohamed et al., 2019;Sahni, Panwar, & Kaur, 2015;Venugopal et al., 2017).The effect of reaction time and plant extract concentration on green synthesis of nanoparticles was analyzed.The absorption spectra exhibited that the SPR peak rises with increasing extract concentration and time.Therefore, high concentration of PAE and CSE avail time dependent optimum amount of bioactive ingredients required to reduce silver ions to silver nanoparticles.Contrary to this, no SPR peak was observed for the highest concentration (16%) of CSE used in this study.
Furthermore, broadening of the SPR peaks and shift from the blue wavelength towards the long wavelength with increasing concentration of the plant extracts was registered.This is attributed to increase in size and change in shape of NPs, decrease in the NPs interspace and agglomeration of NPs in colloidal dielectric solutions (Kelly et al., 2003;Lee & El-Sayed, 2006).This speci es that highly concentrated plant extracts mostly PAE synthesizes clustered and polydispersed nanoparticles.SPR peaks were registered between 260 nm and 300 nm not within the optical range of silver nanoparticles.Shaik et al., (Shaik et al., 2018) reported similar ndings and attributed the SPR bands to absorbance by compounds with benzene and aromatic rings.These bands are associated with π→π* transition (Nasrollahzadeh, Sajadi, Babaei, & Maham, 2015;Nasrollahzadeh, Sajadi, & Khalaj, 2014) and validate the binding of polyphenolics and antioxidant-like compounds with aromatic rings in the plant extracts to the surface of silver nanoparticles (Shaik et al., 2018).
XRD analysis agreed with UV Vis spectroscopy results.Peaks of Ag NPs were displayed in the XRD OriginPro plots ratifying presence of Ag NPs.XRD array showed four strong peaks located at 2θ values of 38.0˚, 44.3˚, 64.5˚, and 77.4˚.It's worth noting that these values are identical for both PAE and CSE green synthesized NPs.These values harmonized well with the cubic crystal lattice planes, ( 111), ( 200), ( 220) and ( 311) respectively of silver metal using International Centre for Diffraction Data (ICDD) database PDF le number 004-0783.However, AgNPs biosynthesized by CSE were of high crystallinity in nature as demonstrated by peaks of higher intensity.Other peaks are corresponding to impurities such as phytochemicals used in the bio-fabrication of AgNPs.
FTIR spectroscopy was employed to pro le the phytochemicals in PAE and CSE used in bio-reduction of silver ions to nano-scale by identi cation of the possible organic functional groups capping the Ag NPs  Morril, 1962).FTIR spectra of PAE and CSE exhibited similar bands within wavenumber ranges (cm − 1 ) of 3650 − 3400, 2960 − 2850, 2349, 2140 − 1990, 1650 − 1550, 1390 − 1370 and 600-800  Similar to these ndings, green tea polyphenols catechins exhibited OH intermolecular bonds linked to methyl functional groups of the different constituent units (Botten, Fugallo, Fraternali, & Molteni, 2015).
FESEM images exhibited that AgNPs green synthesized by different concentration of CSE and PAE were spherical in shape.This is in line with other studies which fabricated AgNPs using plant extracts (Ajitha, Ashok Kumar Reddy, & Sreedhara Reddy, 2015;Gholami, Shahzamani, Marzban, & Lashgarian, 2018;Hamouda, Hussein, Abo-Elmagd, & Bawazir, 2019).The nanoparticles were highly aggregated with average size distribution varying from approximately 3 nm to 19 nm for nanoparticles.Clustering of nanoparticles may be attributed to interaction of concentrated nanoparticles with the organic components of the plant extracts used in biosynthesis.This is supported by appearance of a slimy like substance on the surface and in between nanoparticles especially at high concentrations of plant extracts in FESEM images.The procedure used in the preparation of the lm of nanoparticles for FESEM imaging also in uences the level nanoparticle agglomeration.To characterize the particle size and nanostructure using high resolution scanning electron microscopy (SEM), Chattopadhyay et al., 2013a) recommended a method that prepares lms with disaggregated nanoparticles.The improved method involves suspension of nanoparticles in de-ionized water at a concentration of 1 mg/mL followed sonication to form a homogenous suspension.The homogenous suspension is diluted using a 1-20 dilution factor and then 1 µl of the diluent be is spread on to a carbon tape, dried, gold/carbon coated, and images taken.
Antimicrobial activity of nanoparticles depends on size and shape distribution.Nanoscale materials with minute size possess enhanced chemical reactivity, high diffusion rate and high penetrative power while those with various form distribution offer different modes of antimicrobial activity (Ssekatawa et al, 2020;Simon-Deckers et al. 2009).PAE and CSE mediated biosynthesis attained very small nanoparticles as revealed by FESEM, XRD and DLS analyses.However, only the spherical form was achieved by this study.Zeta potential estimation using the DLS technique revealed that PAE and CSE green synthesized AgNPs were positively charged.This promotes electrostatic interface with the negatively charged bacterial cell walls (Dickson and Koohmaraie 1989).Thus, minute and positively charged nanoparticles possess enhanced antibacterial activity (Lu et al., 2013).
The antibacterial potential of the green synthesized AgNPs on human pathogenic CR E. coli and K. pneumoniae was assessed using the growth inhibitory zone.The NPs demonstrated e cient antibacterial activity with the growth inhibitory zones larger than those achieved in other studies (Raman, Park, Sakthivel, & Suresh, 2017).Furthermore, several antimicrobial bactericide e cacies are dose dependent (McKenzie, 2011).However, high concentrations exhibit nonselective cytotoxicity.Therefore, therapeutic agents with very low MIC and MBC are preferable.This study achieved signi cantly low MIC and MBC of 125/250 µg/ml and 250/500 µg/ml respectively comparable to the recommended NP nontoxic dose (100 µg/mg) to mammalian cells and way below MICs reported by other studies (Jafari et al., 2018;Reithofer, Lakshmanan, Ping, Chin, & Hauser, 2014).The susceptibility pattern of both CR and sensitive E. coli and K. pneumoniae to AgNPs (MIC = 125 µg/ml for E. coli and 250 µg/ml for K. pneumoniae) was identical which is an indication of no mechanism of resistance to AgNPs in carbapenemase producing bacteria.Furthermore, compared to CSE-AgNPs, statistically PAE-AgNPs exhibited smaller zones of bacterial growth inhibition.This may be attributed to higher levels of the slimy PAE materials (proteins and polysaccharides) as revealed by FESEM images that might prevent high rate migration of the nanoparticles within the agar.

Conclusion
Evolution of MDR pathogens presents a challenge to health care systems and calls for speedy research to design alternative therapeutics such as NPs without compromising the safety of consumers.Therefore, this study employed an ecofriendly method the green synthesis to yield AgNPs.   FTIR spectra for biosynthesized AgNPs assigned to O-H intermolecular stretching bonds found in alcohol or phenols, C-H stretching vibrations in methyl groups alkanes, O = C = O stretching bonds of carbon dioxide, N = C = S stretching bonds of isothiocyanate, N-H bending oscillations of primary amines, C-H bending bonds of gem dimethyl groups in alkanes and C-Cl stretching bond of halide alkyl correspondingly.However, Ag NPs biosynthesized by CSE were also capped by unique functional groups with spectra wavenumber ranges (cm − 1 ) of 1390 − 1370 and 1250 − 1020 associated stretching vibrations of O-H group in alcohols or phenols and C-N of amine respectively.Additionally, PAE green synthesized Ag NPs exhibited distinctive organic functional groups within wavenumber series (cm − 1 ) of 4000 − 3700, 1560 − 1500, 1440 − 1395, 1310 − 1250 and 1124 − 1087 associated with free OH probably due to water, N-H stretching vibrations due secondary amine, O-H bending vibrations in carboxylic acid group and C-O stretching vibrations of secondary alcohol respectively.

Table 1 :
Concentration of plant extract, volume of plant extract and volume of silver nitrate used to green synthesize Ag NPs Concentration of Prunas africana extract (%) Volume of plant extract (ml) Volume of Silver nitrate (ml) Total volume(ml) . PAE and CSE mediated photosynthesis of AgNPs was additionally con rmed by transition from light brown to deep brown colour for PAE and dark blue colour for CSE while Silver nitrate solution and plant extracts remained colourless and light brown respectively throughout the experiment, Fig 4.

Table 3 :
AgNPs-CSE and AgNPs-PAE associated organic functional groups as revealed by FTIR analysis

Table 4 :
MIC and MBC values of AgNPs against Carbapenem resistant E. coli and K. pneumoniae.

Table 5 :
Sensitivity tests of AgNPs green synthesized by PAE and CSE extract showing inhibitory zone in millimeters The Camellia sinensis and Prunus africana photosynthesized Ag NPs exhibited potent antibacterial activity against CR clinical isolates DeclarationsEthics and consent participate: Not applicable Consent for publication: Not applicable Funding: