Isolation and identification of Endophytic fungi isolated from Tamarindus indica
The leaves of Tamarindus indica was used as explant and endophytic fungi was isolated as described in the methods. After sequencing of the selected strain, phylogenic tree was constructed using Maximum Likelihood algorithm by aligning closely related sequences and as per sequence similarity and clustering of the phylogenetic tree, the sequence was identified as Penicillium sclerotiorum and deposited in NCBI Genbank with accession number MK942602. Penicillium is one of the widely distributed fungi in environment (Fig.1a).
Penicillium sclerotiorum mediated synthesis of silver nanoparticles (PsNps) and Biophysical characterization
Pure cell free extract of Penicillium sclerotiorum was used for the synthesis of silver nanoparticles and the colour change was noted from pale while to brown colour which indicates the synthesis of Penicillium sclerotiorum mediated synthesis of PsNps (Inlet fig.1 b,c,d). The UV-visible absorption spectrum was taken between 200-800nm, which showed the SPR peak around 400nm which confirmed the synthesis of Penicillium sclerotiorum mediated synthesis of silver nanoparticles (PsNps) (Fig.1 e).
FTIR spectrum of PsNps was taken to observe the possible biomolecules involved in the reduction and capping to achieve the synthesis of stable PsNps. The Peak wavelength (cm-1) of 3146, 2902, 2107, 1992, 1887, 1610, 1311, 1078, 985, 627 correspond to functional groups of carboxylic acid (O-H stretching), N-H stretching amine salt and alcohol (O-H stretching), isothiocyanate N=C=S stretching, aromatic compound (C-H bending), aromatic compoundα,β-unsaturated ketone (C=C stretching), sulfone (S=O stretching), primary alcohol (C-O stretching), alkene (C=C bending), halo compound (C-Br stretching) respectively (Fig.1 f) (Table 1).
The DLS technique was utilized to identify the size of nanoparticles based on Brownian movement and it was observed as 331.2 nm and the negative charge-20.6 mV confirms the stability of the nanoparticles synthesized using Penicillium sclerotiorum extract (Fig.2 a, b).
From the field emission microscopic image at 25.00 KX magnification at 1µm scale, 50.00 KX magnification at 200nm scale and 100.00KX magnification at 100nm scale, the nanoparticles were observed as different shaped and different within nanometer in range (Fig.3 a,b,c). The elemental composition reveals the presence of Ag by showing peak at 3KeV. Apart from silver other elements such as C, O, Mg, Si, S, Ca, Al and Cl were present in the nanoparticles (Fig.3 d). The percentage weight of each element was found as 35.37%, 29.49%, 18.32%, 6.95%, 6.91%, 1.58%, 0.82%, 0.56% for Ag, O, C, Ca, Cl, Si, S, Al respectively. The percentage of atom was found as 7.87%, 44.23%, 36.61%, 4.16%, 4.68%, 1.35%, 0.61%, 0.5% for Ag, O, C, Ca, Cl, Si, S, Al respectively (Fig.3 e). These elements may derive from the secondary metabolites of fungal extract, which act synergistically in imparting efficient antibacterial activity.
Antibacterial potential of PsNps
Many pathogens are developing resistance towards routine antibiotics. Hence, there is a pressing need of the hour to find alternative solution by synthesizing nanoparticles using Penicillium sclerotiorum extract. As Penicillium is a very important genus used for the mass production of valuable products such as penicillin. Penicillium sclerotiorum was reported to produce antimicrobial secondary metabolites (Petit et al., 2009) which was utilized in this study to synthesize silver nanoparticles and validated against E. coli ATCC (25922) and urinary tract infection causing antibiotic resistant E. coli. The minimum inhibitory concentration is the minimum concentration of PsNps which inhibit the growth of test organism. The visual turbidity of E. coli ATCC (25922) and UTI causing clinical pathogen was observed and the wells with invisible growth was considered as MIC. The MIC of E. coli ATCC (25922) and UTI causing MDR pathogen was calculated as 0.75 µg/ml and 6.25 µg/ml respectively. The decrease in the growth rate of bacterium at their MIC concentration was represented graphically in fig. 4 a. The minimum bactericidal concentration is the lowest concentration of PsNps which completely kills the test organism. The MBC of E. coli ATCC (25922) and UTI causing MDR pathogen was calculated as 6.25µg/ml and 12.5µg/ml respectively (Table 2). This study focused on assay to validate the antibiofilm effect of PsNps on biofilm forming UTI causing E. coli. From our experiments it was observed that upon treatment with 12.5 µg/ml of PsNps, the biofilm formation was reduced by 87% and 85% for E. coli ATCC (25922) and UTI causing MDR E. coli pathogen (Fig.4 b). In addition, this research work focused on studying the amplification of CTX-M-15 gene upon treatment with PsNps and ampicillin. From the results it was observed that the expression of CTX-M-15 gene was found in control and ampicillin treated strain, however the expression of CTX-M-15 gene in PsNps treatment was suppressed (Fig.4 c).