Antimicrobial & anticancer activities of extracellular silver nanoparticles biosynthesised by Haloferax sp SNP6

Sampling and site description

Samples of brine and sediment were sampled from a solar salt pan located on Jeddah’s southern beach (coordinates: 21°10′16.04″N, 39°11′5.94″E), Saudi Arabia, from October 2023 to April 2024. Initial microbiological isolation and primary screening were conducted.

Isolation and cultivation conditions

Haloarchaea were isolated from brine and sediment samples using the method as described previously¹⁰. This method involves using NTYE (25% NaCl, 2% MgSO₄.7H₂O, 0.5% KCl, 0.5% tryptone, 0.3% yeast extract, and 2% agar). The pure strains were acquired by repeatedly culturing and preserving them on NTYE.

Screening for Haloarchaeal strains resistant to silver

Following a two wk incubation period at 40°C, five distinct Haloarchaeal isolates were successfully obtained¹⁰. The isolates’ ability to thrive in AgNO₃ concentrations from 0.05 to 1 mM was examined. The cell culture was analysed using a UV-spectrophotometer (UV-2600 Series, SHIMADZU) at 600 nm. The medium lacking AgNO₃ served as a negative control, while non-inoculated media with AgNO₃ served as a positive control.

Extracellular synthesis of silver nitrate NPs

The chosen Haloarchaeal strain SNP6 was cultured using the NTYE, supplemented with 0.5 mM AgNO₃. The transition of colour from red to brown¹¹ evidenced the visual observation of NP biosynthesis. The supernatant with AgNPs was concentrated by spinning through an Ultra-15 Centrifugal Millipore membrane (Amicon) with a 3,000 Da cutoff for 10 min at 4°C at 14,000 rpm. To collect the NPs, they were dried on a glass slide, rinsed with one per cent glacial acetic acid, and scraped into a powder.

Identification of the isolates

The Haloferax sp strain SNP6 genomic DNA was isolated using a modified method¹². The 16S rRNA gene was amplified utilising universal primers specific to Archaea (Invitrogen, USA): 5’-ATT CCG GTT GAT CCT GCC GG-3’ (positions 6-25) and 5’-AGG AGG TGA TCC AGC CGC AG-3’ (positions 1540-1521)¹³. PCR was performed under standard thermal cycling conditions: 30 cycles of 95°C for 5 min (pre-denaturation), 94°C for 1 min (denaturation), 60°C for 1 min (annealing), and 72°C for 1.5 min (extension), followed by a final elongation at 72°C for 10 min. PCR products were sequenced by MacroGen (Korea). Strain identification was done using NCBI BLAST, and phylogenetic analysis was conducted using MEGA11 software.

Characterisation of extracellular synthetised AgNPs

The UV-2600 Series spectrophotometer (SHIMADZU) was employed to determine the extinction coefficient of the colloidal solution of Hal-AgNPs. Transmission electron microscopy (TEM) was used to assess the morphology and size of Hal-AgNPs, utilising the HF-3300 model from Hitachi High-Tech Canada, Inc. A second analysis of the Hal-AgNP size distributions was conducted with ImageJ, a free programme (Version 1.53d) downloaded from the NIH website ( http://rsb.info.nih.gov/ij ). A Malvern analytical X-ray diffractometer (XRD) was employed for structural characterisation. The active groups were identified using a Perkin-Elmer Spectrum GX spectrometer and Fourier-transform infrared spectroscopy (FTIR).

Antimicrobial activity of Hal-AgNPs

The antimicrobial activity of Hal-AgNPs was assessed using the agar well-diffusion method by the guidelines outlined by Espinel-Ingroff et al¹⁴. This method was employed against six microbial strains: Escherichia coli (ATCC 8739), Staphylococcus aureus (ATCC 6538), Bacillus subtilis (ATCC 6633), Pseudomonas aeruginosa (ATCC 90274), Aspergillus niger, and Candida albicans (ATCC 10221). Wells were loaded with 50 µL of Hal-AgNPs, with gentamicin as the positive control for bacterial strains and fluconazole for fungal strains. All experiments were conducted in triplicate to ensure data accuracy and reproducibility.

Determination of Minimal-Inhibitory-Concentration (MIC)

MIC of Hal-AgNPs was assessed for microbial strains showing positive results. Hal-AgNPs were diluted to concentrations (31.25, 62.5, 125, 250, 500, and 1000 μg/mL) and mixed with fresh microbial colonies (10⁶ to 10⁸ CFU/mL). The tubes were incubated for 24 h at 37°C with a control. The MIC endpoint was determined as the lowest concentration showing no visible growth. Tube turbidity was recorded before and after incubation to validate the MIC value.

Haemolytic action of Hal-AgNPs

Haemolytic activity of Hal-AgNPs was assessed by incubating human red blood cells (RBCs) from healthy donors with NP concentrations ranging from 50 to 1000 μg/mL. RBCs were washed and resuspended in phosphate buffer (pH 7.4), mixed with Hal-AgNPs, and incubated at 37°C for 1 h. Following centrifugation, the absorbance of the supernatant was measured at 541 nm. Haemolysis percentage was calculated using the formula:

$\text{Haemolysis}\left(\%\right)=\left[\left(\text{Abs sample}-\text{Abs blank}\right)/\text{Abs positive control}\right]\times \text{1}00.$

Deionised water and PBS were used as positive and negative controls, respectively.

Cytotoxicity of Hal-AgNPs on cancer cells

The cytotoxicity of Hal-AgNPs was evaluated against human breast a nd cervical cancer cell lines, as well as non-cancerous Wi38 cells, in accordance with Hon et al¹⁵. Cells were cultured and treated with serial dilutions of Hal-AgNPs prepared in RPMI medium with 2 per cent serum. Following incubation, cell viability was assessed using the MTT assay. Absorbance was measured spectrophotometrically, and cytotoxic effects were quantified by comparing the treated and control. IC₅₀ values were calculated to determine the inhibitory concentration of Hal-AgNPs.

Statistical analysis

This study evaluated the biological activities of extracellular Hal-AgNPs strain SNP6 using two-tailed Independent Samples t-tests, conducted via SPSS (Version 26.0, IBM Corp.). Cytotoxicity was assessed on human cancer cell lines, compared with non-cancerous Wi38 fibroblasts. The two-tailed approach was employed to detect differences in either direction (increase or decrease) in cell viability and bacterial growth inhibition between the treatment and control groups, with P values of < 0.05 considered statistically significant. Data were expressed as mean±standard deviation (SD).

Biosynthesis of extracellular AgNPs

The nanoparticle formation is typically indicated based on monitoring the colour change following which the synthesis is confirmed. In this study, after incubating the culture supernatant of Haloferax sp strain SNP6 with AgNO₃ (0.5 mM) at 37°C for one week, the colour changed from orange-red to brown-black. This change occurred when secondary metabolites from Haloferax sp strain SNP6 were mixed with a solution of silver nitrate. The control (culture media without AgNO₃) showed no colour change. The existence of a brown colour demonstrated the Ag NPs formation.

Archaeal strain identification

The 16S rRNA gene sequencing examination showed that the archaeal strain belongs to the genus Haloferax and is more specifically selected as Haloferax sp The sequence analysis reveals a similarity of approximately 90 per cent with known sequences. The 16S rRNA gene sequences for this Haloarchaea strain have been formally deposited in GenBank databases and are recorded with the accession number PP504131 (Fig. 1).

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Fig. 1.

Neighbour-Joining phylogenetic tree was constructed using 16S rRNA gene sequences to illustrate the relationship between Haloferax sp strain SNP6 and closely related taxa obtained from the NCBI database. The tree was generated using MEGA X software (Version 10.1.8). The scale bar represents a rate of 1 substitutions per nucleotide location.

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Characterisation of synthesised Ag NPs

The biosynthesised Hal-AgNPs exhibited semi-spherical morphology with sizes ranging from 2 to 14 nm, as observed by TEM, and were embedded in organic sheaths (Fig. 2). FTIR analysis revealed prominent absorption bands at 3453, 1634, 1111, and 603 cm⁻¹, indicating the presence of functional groups that enhance nanoparticle stability. XRD analysis showed distinct peaks at 37.85°, 41.13°, 61.49°, and 74.23°, corresponding to the (111, 200, 220, and 311) planes, respectively, confirming the crystalline nature of the Hal-AgNPs (Fig. 3).

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Fig. 2.

(A) The histogram illustrates the distribution of particle diameter sizes of the Hal-AgNPs, which were determined through analysis of TEM images. The red line represents a Gaussian distribution function. (B) TEM images of Hal-AgNPs (scale bar = 100 nm).

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Fig. 3.

(A) FTIR spectrum of Hal-AgNPs strain SNP6. (B) XRD AgNPs Hal-AgNPs strain SNP6.

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Antimicrobial activity of Hal-AgNPs

Figure 4 shows a comparative analysis using an independent t-test to assess the antimicrobial efficacy of Hal-AgNPs and the standard drug against six pathogenic microbial strains. The results suggest that the antimicrobial efficacy of Hal-AgNPs closely resembles that of the reference medication. Regarding B. subtilis, the average inhibition zone for Hal-AgNPs (28±1.7 mm) was slightly more significant than that for gentamicin (26±1.3 mm). S. aureus exhibited similar inhibition zones for Hal-AgNPs (18±1.2 mm) and gentamicin (16±1.0 mm). P. aeruginosa exhibited a larger average inhibitory zone for Hal-AgNPs (24±1.5 mm) than gentamicin (23±1.2 mm). E. coli showed similar inhibitory zones for Hal-AgNPs (18± 1.1mm) and gentamicin (17.0±1.2mm). C. albicans showed a somewhat larger zone of inhibition when exposed to Hal-AgNPs (27±2 mm) compared to Fluconazole (25±1.1 mm). Additionally, A. niger did not display any activity towards Hal-AgNPs (NA), whereas Fluconazole demonstrated a mean inhibition zone of 17±0.9. The MIC was utilised to evaluate the growth inhibitory effect of Hal-AgNPs against both bacterial and fungal species (Fig. 5). MIC values of Hal-AgNPs were recorded as 250, 62.5, 125, and 500 μg/ mL for E.coli, B. subtilis, P. aeruginosa, S. aureus, and C. albicans, respectively.

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Fig. 4.

Antimicrobial activity of Hal-AgNPs against pathogenic microbial strains.

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Fig. 5.

MIC values for Hal-AgNPs against pathogenic microorganisms.

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The cytotoxicity of extracellular Hal-AgNPs produced by Haloferax sp strain SNP6 was assessed by evaluating their haemolytic activity on RBC at different doses. The findings revealed a direct correlation between the dosage and the level of haemolytic activity, with the highest concentration of 1000 µg/mL resulting in 4.5 per cent haemolysis. Hal-AgNPs demonstrated minimal haemolytic activity across all tested concentrations, with haemolysis consistently below 5 per cent. Notably, no haemolysis was observed at 50 µg/mL, and only 2.2 per cent at the highest dose (800 µg/mL), indicating low cytotoxicity and good biocompatibility (Fig. 6).

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Fig. 6.

Haemolytic action of Hal-AgNPs.

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The cytotoxic effects of Hal-AgNPs on various human cell lines, revealed distinct sensitivity patterns across cancerous and non-cancerous cells (Fig. 7). For cancer cell lines, MDA-MB231 and Mcf7 showed a high level of sensitivity to Hal-AgNPs, with IC₅₀ values determined at 113.3 µg/mL±0.59 and 79.1 µg/mL ±0.9, respectively. This indicated a potent cytotoxic effect of Hal-AgNPs at relatively low concentrations. Similarly, the HELA cell line (cervical cancer) exhibited significant sensitivity, with an IC₅₀ of 83.56 µg/mL±1.2. Conversely, the non-cancerous human cell line Wi38 demonstrated considerably higher NP resistance, with an IC₅₀ value of 218.1 µg/mL ± 3.5 suggesting that Hal-AgNPs have lower toxicity towards normal cells at the tested concentrations. The order of sensitivity, from most to least affected, was observed as follows: Mcf7 > HELA > MDA-MB231, with non-cancerous Wi38 cells showing the least sensitivity.

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Fig. 7.

Cell viability percentage of (A) MDA-MB-231, (B) MCF7, and (C) HeLa cells compared with Wi38 Cell Line (non-cancerous human cell line), treated with Hal-AgNPs.

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