Fabrication And Optimization of Bio-silver Nanoparticles: Activity Evaluation Against Beta-lactamases-Resistant Enterococcus Faecalis

Due to the presence of antibiotic-resistant genes, treatment options of clinical isolates are exceedingly limited. This study was aimed to fabricate, optimize, characterize, and evaluate the action of silver nanoparticles (AgNPs) against a clinical isolate of Enterococcus faecalis. A combination of cell-free supernatant (C-FS) of the �lamentous fungus Fusarium solani and Gram-negative Comamonas aquatica for AgNPs formation was proposed; the antigrowth and antibio�lm of AgNPs against E. faecalis harboring bla TEM and bla CTX-M genes were assessed. The ratio of 1:2 v/v (C-FS:AgNO 3 ) at pH 9.0 for 72 h in 1 mM AgNO 3 were the optimal conditions for AgNPs formation. UV-vis absorption peak appeared at 425 nm and the crystalline nature of synthesized particles was veri�ed by X-ray diffraction (XRD). Fourier transform infrared spectroscopy (FTIR) analysis con�rmed the interaction of protein molecules with the AgNPs. Transmission electron microscopy (TEM) analysis demonstrated that fabricated AgNPs were relatively monodispersed, approximately spherical, and of size 2-7.5 nm. bla TEM and bla CTX-M were detected in E. faecalis; the growth and bio�lm of E. faecalis were signi�cantly decreased by the action of 12.5 µg/mL AgNPs. This is the �rst study proposing alternative sources to form AgNPs via synergistic metabolites of F. solani and C. aquatica. The results here offer a foundation for developing an effective therapy using AgNPs against clinical pathogens.


Introduction
In the last decade, the rate of antibiotic resistance has considerably elevated as pathogens evolve a variety of mechanisms for resistance to antibiotics [1].As a phenomenon, antibiotic resistance is often associated with infection and is therefore also related to virulence, as in the cases of bio lm-producing bacteria [2].The latter is the cause of several acute and chronic human infections such as the native valve endocarditis, pneumonia in cystic brosis patients, infection of chronic wounds, catheterassociated infections, and chronic bacterial prostatitis [2,3].Most of the currently used antibiotics are becoming ine cient against the bio lm-associated and multidrug resistant (MDR) microorganisms.
Therefore, it is necessary to bioprospecting for novel solutions to mitigate such microbes.[4].Enterococcus faecalis is one of the most common primary infectious enterococcal species identi ed in hospital-acquired infections [5].E. faecalis is a Gram-positive bacterium and is ranking among the most common nosocomial pathogens isolated from the bloodstream, surgical sites, and urinary tract infections [6].It is also involved in clinical pathogenesis such as wound infections, infective endocarditis, meningitis, and endodontic infections [7].E. faecalis has the ability to produce a bio lm composed mainly of speci c cell surface protein (Esp), where the lm plays a vital role in the adherence mechanism to surfaces, especially of medical devices in chronicle infected patients [8].
Silver nanoparticles (AgNPs) are an alternative to combating bacterial bio lms.AgNPs possess e cient antimicrobial and anticancer activities [9].Also, they are used in wound repair [10] and the promotion of bone healing [11].Silver shows less toxicity towards humans at lower concentrations; thus, it has been widely incorporated in drugs under various forms such as salts, immobilized ions, or nanoparticles [12].
The exact mechanism of AgNPs against pathogens is still not clear; however, there are various proposed mechanisms of action including disturbance of the cell membrane, alteration of cellular DNA and proteins, respiratory chain blockage, or the generation of reactive oxygen species [13].Fabrication of stable AgNPs is a very challenging task; physical methods have low yields, while the chemical ones cause harmful effects on the environment due to use of toxic solvents and the regeneration of hazardous byproducts which can limit the use of AgNPs for clinical applications [14].Biogenic methods for the synthesis of NPs are a strong alternative [15].Qurbani and Hamzah [16] found that the lamentous fungus Fusarium solani and Gram-negative Comamonas aquatica grow well in co-culture and tolerate high concentrations of metals; the study reveals synergistic relationships between F. solani and C. aquatica that robustly remove metals in the culture medium [16].Here, we examined how the supernatants of F. solani and C. aquatica, cooperatively, can be used to synthesize AgNPs in vitro.The effect of AgNPs against a common clinical isolate of infection-derived E. faecalis was also studied.

Materials And Methods
Source of microorganisms F. solani and C. aquatica were used as a source for nanoparticles synthesis.Both organisms were previously isolated and identi ed by Qurbani and Hamzah [16].For the antibacterial experiment, E. faecalis was selected as a model.E. faecalis is a clinical bacterium isolated from a urine sample in Sulaymaniyah Teaching Hospital.E. faecalis was identi ed via VITEK2 (BioMerieux, USA) instrument using a Gram-positive VITEK2 ID card.

Preparation and optimization of bio-AgNPs
Fresh cultures of F. solani and C. aquatica were grown separately in 250 mL Erlenmeyer asks containing 100 mL of nutrient broth.The asks were incubated at 30°C for 48 (for the bacterium) and 72 h (for the fungus).C-FS from the culture asks were collected by centrifugation (Biofuge Stratos, Germany) at 5,000 × g for 20 min at 4°C.For AgNPs fabrication, an equal amount of C-FS of both organisms were mixed with 1 mM of lter-sterilized AgNO 3 solution (Sigma, USA, purity 99.9%) as a nal concentration.
The ask was kept for 72 h at room temperature under static conditions.AgNPs were collected at 14,000 × g for 20 min at 4°C (Mikro 200R, UK) and were washed three times with sterile distilled water.The obtained precipitate was kept in a Petri dish and left in the oven for drying at 40°C for 24 h (WT`C Binder, Germany).The dried AgNPs were scraped out and obtained in powder form for further study [17].The effects of four variables on the product yield of AgNPs were optimized by varying one parameter at a time, including AgNO 3 concentration (0.5, 0.75 and 1.0 mM), pH values (5.0, 7.0, 9.0, and 11.0), reduction time (24,48, and 72 h) and volume ratio (1:1, 1:2, 2:1 v/v C-FS:AgNO 3 ).All experiments were carried out in triplicates.

Characterization of bio-AgNPs
The bio-reduction of Ag + ions was monitored with UV-vis spectrum and was recorded with UV-vis spectrophotometer (Kary60, USA) at a resolution of 2 nm within a range of A 300 to A 800 nm.FTIR analysis was carried out in the range of 400-4,000 cm− 1 at a resolution of 4 cm−1 (PerkinElmer 1600, USA).Crystal structure and size of the AgNPs were investigated by X-ray diffraction using an X 'Pert Pro diffractometer (Pan Analytical, Netherlands) equipped with Cu-K-α radiation (1.5406 Å), at the xed operating voltage and current of 45 kV and 40 mA, respectively.The shape and size of the AgNPs were further determined by TEM (PHILIPS model CM120, Netherlands).

Antibacterial activity of bio-AgNPs
For the agar well diffusion assay, E. faecalis was grown overnight in Luria-Bertani broth (LB) at 37°C [18].CFU/mL was adjusted to be 10 8 .Then, 100 µL culture broth was spread evenly on Müeller-Hinton agar plates.Wells were made using gel puncture, and 100 µL of different concentrations of AgNPs (12.5, 25, 50, 75, and 100 µg/mL) were loaded into certain wells.Afterward, the plates were incubated at 37°C for 24 h, then the zone of inhibition was measured (mm).
Minimum inhibitory concentration (MIC) and antibio lm activities of AgNPs were conducted according to [19] with a few modi cations.Brie y, 120 µL of bacterial culture (10 8 CFU/mL) were placed in a 96-well microtiter plate.Then, 80 µL of different concentrations of AgNPs (3, 6, 9, 12.5, 25, 50, 75, and 100 µg/mL) was added.LB broth (200 µL) was used as a negative control.The plate was then incubated for 24 h at 37°C under gentle shaking in microplate incubator-shaker PST60 HL Plus (BOECO, Germany).The absorbance of each well was measured at 600 nm using a microtiter ELISA reader (Biotech µQuant, USA).Growth percentage was calculated based on the average and standard deviation of triplicate results.
Besides, 5 µL was taken from each well and spotted onto LB agar plates.Simultaneously, 5 µL was also spotted on Congo Red Agar (CRA) plates (for the bio lm experiment).The plates were then incubated at 37°C for 18 h.
Regarding the antibio lm activity of AgNPs, the contents in the microtiter plate wells were discarded and washed three times with 200 µL phosphate buffer saline (PBS, pH 7.2).After drying, the remained wellattached cells were stained with crystal violet (0.1%) for 30 min.The wells were then washed 3 times with distilled water after removing the excess stain.The plate was then allowed to dry at room temperature after which 200 µL ethanol (95%) was added to the wells to solubilize the stain.Then, the wells were read at 595 nm using a microplate reader (Biotech µQuant, USA).Optical density (OD) readings were then converted to percent of inhibition (%).This experiment was performed 3 times to compare and analyze the average of each result.
Protein leakage and detection of bla TEM and bla CTX-M genes in E. faecalis Protein leakage from E. faecalis cells was determined by the A280 assay [20] using NanoDrop 2000 (ThermoFisher, USA).Brie y, bacterial suspension with an OD of about 0.2 at 600 nm (~10 8 CFU/mL) was prepared in LB and treated with 9 µg/mL AgNPs.After overnight at 37°C, samples were centrifuged at 14,000 × g for 5 min at 4°C using Mikro 200R centrifuge (Hettich, UK).Afterward, the collected supernatants were subjected to protein quanti cation.On the other hand, total genomic DNA from untreated and treated cells was extracted using Presto™ Mini gDNA Bacteria Kit (Geneaid, Italy) following the manufacture's guidelines.PCR was performed via ThermoCycler T100TM (Bio-Rad, Singapore).The sequences of the selected primers are shown in Table 1 [20]; the primers were designed to amplify partial sequences of bla TEM and bla CTX−M genes in Gram-negative Klebsiella pneumoniae.PCR reaction mixture setup contained 10 µL master mix, 1 µL of forward and reverse primers each, 1 µL of DNA, and 7 µL of distilled water giving a total of 20 µL.The PCR protocol for 30 cycles was as follows: initial denaturation at 94°C for 2 min, then 94°C for 30 s, 55°C for 30 s, 68°C for another 30 s, followed by a nal extension at 68°C for 7 min.The PCR products were analyzed by electrophoresis using 1% agarose gel in TAE buffer at 90 V for about 40 min, stained with ethidium bromide, and the image was captured via MultiDoc-It™ Imaging System (UVP, USA).

Optimization and characterization of bio-AgNPs
In the current study, optimal formation of AgNPs was studied to achieve, primarily, good mono-dispersity, stability, better yield, and biocompatibility of the particles at 25°C under light conditions.Initially, the extracellular biogenic synthesis of AgNPs was con rmed through the visual color change in the reaction mixture as well as via UV-vis spectroscopy.The intensity of the solution, gradually, changed from pale yellow to dark brown after about 18 h of incubation.Moreover, variation in reaction conditions such as AgNO 3 concentrations (Fig. S1, in supplementary le), pH values (Fig. S2, in supplementary le), volume ratios of C-FS with AgNO 3 solution (Fig. S3, in supplementary le), and reduction time (Fig. S4, in supplementary le) directly affect the synthesis.The best conditions for AgNPs fabrication (Fig. 1) were at pH 9.0 for 72 h in 1 mM of AgNO 3 using 1:2 v/v (C-FS:AgNO 3 ).
FTIR analysis was carried out to identify the interactions between the biomolecules and nanoparticles (Fig. 2A).Spectrum reveals the presence of eight distinct peaks at 1056, 1409, 1462, 1597, 1643, 2987, 3086, and 3279 cm-1 in the region of 400-4000cm-1.The peaks at 3279 cm-1, 3086 cm-1, and 2987 cm-1 indicate the presence of alcohol O-Hstr, primary amine N-Hstr, and alkaline C-Hstr vibration of protein, respectively.The peaks at 1643 cm-1 and 1597 cm-1 represent the C=O carbonyl group which refers to amide of protein.The other bands lie at 1462 cm-1, 1409 cm -1, and 1056 cm-1 depict the characteristic of aliphatic and aromatic organic compounds that contain C-Nstr vibration.
The crystalline structure of the AgNPs was investigated by the XRD technique.The XRD pattern (Fig. 2B) shows ve characteristic peaks at 2θ values of 27.84°, 32.25°, 46.26°, 54.85° and 57.52°, which can be assigned the planes of ( 111), ( 200), ( 220), (311), and (222), respectively.The average estimated particle size of our sample was 16.46 nm obtained from the full width at half maximum (FWHM) of the peak corresponding to 200 planes by using the Debye-Scherrer equation.
The data obtained from TEM images show that AgNPs have mostly spherical in shape, 2-5 nm in size, and relatively monodispersed, with few agglomerated particles (Fig. 2C).The particle size distribution curve shows that the range of AgNPs size is 2 to 7.5 nm with an average diameter of 4.5 nm (Fig. 2D).

Action of AgNPs against E. faecalis
An inhibition zone was observed against E. faecalis (Fig. S5, in supplementary le).To detect the lowest concentration that completely inhibits E. faecalis visible growth, the MIC was determined (Fig. 3A).The MIC of AgNPs against E. faecalis is 12.5 µg/mL (p-value < 0.0001).Additionally, AgNPs reduced the bio lm formation of E. faecalis by 96 %.Notably, changes in the colony and its surrounding colors on CRA in the presence of nanoparticles indicate that AgNPs have a signi cant role as antibio lm against E. faecalis (Fig. 3A inset).PCR con rmed the presence of bla CTX−M and bla TEM in E. faecalis.Protein leakage was also measured in the supernatant after treating the cells with 9 µg/mL AgNPs.Protein concentration was greatly elevated (7.8 mg.mL −1 ) in comparison with untreated cells (4.7 mg.mL −1 ).

Discussion
The current paper is the rst study investigating the use of C-FS of F. solani and C. aquatica collaboratively as biocatalysts for making AgNPs, shedding light on the metabolite-metabolite interactions between both organisms.Furthermore, the present work investigates, for the rst time, the role of C. aquatica metabolites in AgNPs fabrication.AgNPs absorbed light at different wavelengths and were excited due to charge density at the interface between conductor and insulator; the solution developed a dark brown color within a few hours.Apparently, the combination of metabolites in our C-FS provides a synergistic effect for stabilizing AgNPs leading to the excitation of surface plasmon vibrations.To increase the yield and stability of AgNPs, pH value, reduction time, C-FS with AgNO 3 ratio, and AgNO 3 molarity were studied.Controlling pH values can be used to control certain characteristics of the nanoparticles; pH 9.0 was found to be the optimal value for the maximum absorbance at 438 nm.The bioactive metabolites of the fungal-bacterial supernatant seem to be more stable and possess higher catalytic activity at alkaline pH.In acidic pH value, AgNPs aggregation was observed.Whereas at pH 7.0, there was less synthesis of NPs (data not shown).Multiple studies reported increased nano-production at alkaline pH, corroborating our result [21][22][23].Longer time periods also elevate nanoparticle production [24].
Consistent with most of the literature where 1 mM of AgNO 3 is usually used for AgNPs synthesis, although different concentrations of silver nitrate were applied, 1 mM was found to be the best.The concentration of AgNO 3 is a key parameter that greatly affects the synthesis process; however, very few reports studying metal concentrations for AgNPs biogenic synthesis are available.As the concentration of AgNO 3 decreased to 0.75 and 0.5 mM, the production of AgNPs decreased.Ma et al. [23] reported that a concentration of 2.0 mM AgNO 3 triggered the maximum production of AgNPs, with the absorbance peak at 415 nm.Moreover, AbdelRahim et al. [15] found that the addition of excess metal ions created very large nanoparticles with irregular morphology.
The excreted metabolites by the fungus and bacterium have a strong a nity to bind to the AgNPs surface through free amino groups, cysteine residues, or electrostatic interaction of negatively charged carboxylate groups.Moreover, the metabolites in the C-FS were not only playing roles in the reduction and capping processes but also stabilizing AgNPs.Intriguingly, a previous article examined the importance of C. aquatica in reducing the toxicity of metals and stimulating the growth of F. solani [16].Here, we propose a coupled activity of C. aquatica and F. solani metabolite that recapitulates the reported synergistic activation for AgNPs fabrication.Consequently, more data should be necessary as it might reveal the functional principles of C. aquatica and F. solani metabolites in the synthesis process and, perhaps, shed light on the reduction mechanisms.
Con rming the exact nature of the formed AgNPs, the XRD technique was conducted.As mentioned above, the XRD pattern shows some distinct peaks at 2θ values.All the re ection planes are matched and consistent with the face-centered cubic (fcc) phase of the pure crystalline silver structure's database of the Joint Committee on Powder Diffraction Standards (JCPDS).A possible reason for the variation in the average particle size might be due to the aggregation during the drying process [24].As shown in TEM image, AgNPs with an average size ranging from 2-7.5 nm were obtained.Comparably, AgNPs with a size ranging 6-53 nm have been synthesized from C. acidovorans with spherical, oval, and irregular shapes with a smooth surface [17].
With that in mind, how do AgNPs, for the rst time against E. faecalis, perform?AgNPs inhibited the growth of E. coli, P. aeruginosa, S. aureus, S. enterica, and 7 more clinical isolates of E. faecalis (data not shown here).Our AgNPs, based on the MIC, are able to inhibit the growth and the bio lm of E. faecalis even at lowest concentrations.This might be enhancing the permeability of the cell membrane, formation of free radicals, and interaction with thiol groups, affect cellular signaling (data not shown), reduction of bio lm and DNA intensity.Several mechanisms have been proposed for the bactericidal activity of AgNPs; however, the exact mechanisms remain unclear.Most likely, smaller nanoparticles have greater antimicrobial effects [25,26] and spherical AgNPs show a larger surface area to volume ratio [27].The combination of both these properties might present stronger bactericidal activity.One of the most accepted mechanisms is that the direct contact of AgNPs with large surface areas on a bacterial cell wall could lead to produce pits, resulting in the leakage of cellular contents and, eventually, cell death [28].In certain cases, small nanoparticles of size less than 10 nm particularly, can penetrate the cytoplasm and damage the respiratory chain enzyme thus, causing damage to proteins, reducing transcriptome and inducing cell death [29,30].As such, the cell membrane of E. faecalis was disrupted by the action of AgNPs, and it was clearly supported by the resultant protein content in the supernatant.This indicates that AgNPs could increase permeability and affect membrane transport due to the serious damage of cell membrane structure.Chen et al. [31] reported that AgNPs not only condense DNA, but also combine and coagulate with the cytoplasm of damaged bacteria, resulting in the leakage of the cytoplasmic component.
The current study has been designed to assess the mode of action against E. faecalis, and the results presented here are promising and warrant further investigation.Future studies aimed at assessing and producing clinically feasible sources of AgNPs for in vivo studies are necessary to translate these ndings into clinical use.The correlation between the production of -lactamases and the spread of resistance among isolates of Gram-positive pathogens is very high, forming serious clinical challenges [32].E. faecalis has the propensity to acquire resistance determinants via horizontal gene transfer, and it has shown the frequent occurrence of antimicrobial resistance, especially to tetracycline and erythromycin [33].In this context, this is the rst study to shed light on the existence of bla TEM and bla CTX−M in a clinical isolate of E. faecalis using primers designed to amplify them in Gram-negative bacteria.Probeing the unusual event based on sequence changes of bla TEM and bla CTX−M in E. faecalis might lead to effective prevention strategies and control horizontal nosocomial transmission of organisms.
In this study, the C-FS combination of F. solani and C. aquatica showed synergistic effects for AgNPs synthesis.Optimization studies con rm that pH 9.0 for 72 h in 1 mM of AgNO 3 using 1:2 v/v (C-FS:AgNO 3 ) were the best conditions for AgNPs formation.Moreover, the small size of AgNPs and spherical shape suggests that they are stable particles.The different behavior of AgNPs against E. faecalis has been noticed.Protein leakage suggests that AgNPs might disrupt the cell wall and interfere with cellular components of E. faecalis.The chance of acquiring resistance genes in clinical isolates calls for an effective remedy in the control and surveillance of antibiotic resistance.Although the obtained AgNPs show promising antibacterial agents, further research is strongly recommended to investigate the safe usage of AgNPs.

Supplementary Files
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Table 1
Primer sequences for detection of bla CTX−M and bla TEM -values were obtained (Unpaired t-test) by using GraphPad Prism 8.0.1 software Inc, CA, USA.Statistical signi cance was de ned when the P-value was less than 0.05. P