The current paper is the first 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 first 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 AgNO3 ratio, and AgNO3 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 [21–23]. Longer time periods also elevate nanoparticle production .
Consistent with most of the literature where 1 mM of AgNO3 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 AgNO3 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 AgNO3 decreased to 0.75 and 0.5 mM, the production of AgNPs decreased. Ma et al.  reported that a concentration of 2.0 mM AgNO3 triggered the maximum production of AgNPs, with the absorbance peak at 415 nm. Moreover, AbdelRahim et al.  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 affinity 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 . 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.
Confirming 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 reflection 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 . 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 .
With that in mind, how do AgNPs, for the first 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 biofilm 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 biofilm 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 . 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 . 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.  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 findings into clinical use. The correlation between the production of 𝛽-lactamases and the spread of resistance among isolates of Gram-positive pathogens is very high, forming serious clinical challenges . 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 . In this context, this is the first study to shed light on the existence of blaTEM and blaCTX−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 blaTEM and blaCTX−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 confirm that pH 9.0 for 72 h in 1 mM of AgNO3 using 1:2 v/v (C-FS:AgNO3) 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.