Collection of the plant sample

Fresh and mature garlic roots, leaves, and cloves were carefully cut out and harvested from agricultural land in El-Menofia governorate, Egypt (30° 38′ 40.9″ N 30° 56′ 49.9″ E) in April 2021. The samples were collected in sterilized polythene zip-lock bags and promptly delivered to the botany department’s laboratory at Al-Azhar University in Cairo, where they were preserved at 4 °C in a refrigerator till usage within 24 h after collection.

Isolation of endophytic bacteriaSurface sterilization

The collected garlic (roots, leaves, and cloves) was cleaned thoroughly under running tap water to eliminate dust, and soil remains from the plant surface, and air dried. Then, the plant parts were sterilized by being submerged for 1 min in sterile distilled water, then dipped in ethanol 70% for 1 min, followed by being washed in 2.5% sodium hypochlorite for 4 min, ethanol 70% for 30 s, and lastly rinsed three times in sterile distilled water. To evaluate the effectiveness of the plant surface sterilization, distilled water from the final washing was spread onto a nutrient agar plate, followed by incubation for 24 h at 37 ºC. The efficacy of the plant surface sterilizing procedure has been verified by the lack of microbial colonies on the incubated plates (Ismail et al. 2021; Stelmasiewicz et al. 2023).

Media for isolation of endophytic bacteria

Nutrient agar was utilized to isolate endophytic bacteria. Since the nutrient agar medium cannot suppress the endophytic fungi growth, it was treated with nystatin (30 µg/mL) to suppress the fungal growth.

Isolation and purification of endophytic bacteria

After proper drying of surface sterilized garlic parts (leaves, roots, and cloves), a sterile scalpel was used to cut some of these parts into small pieces 5 mm long in a laminar airflow cabinet and each piece was spread on a surface of the prepared nutrient agar plate. Other parts were ground in a sterile saline solution using a sterile pestle and mortar in the laminar airflow cabinet. About 1 mL of the ground sample was diluted, and a sterile glass -rod was used to spread 0.1 mL of 10− 2 diluted solution onto the nutrient agar plates. All plates were then incubated for 24–72 h at a temperature of 37 ºC (Ismail et al. 2021). After 72 h, bacterial growth was observed on the plates. To obtain pure bacterial isolates, a sterile inoculation loop was used to collect a single colony from each plate and streaked onto fresh nutrient agar plates then incubated at 37 ºC. Then all different isolates were sub-cultured into nutrient broth with 50% glycerol and then kept in a freezer at -80 °C till further experimental use (Sharma and Mallubhotla 2022).

Preliminary identification of endophytic bacterial isolates

All endophytic bacterial isolates were identified through morphological standards, including colony shape and color, as well as microscopic characterization like Gram staining and biochemical tests.

Production of bioactive metabolites

Pure bacterial endophyte isolates were pre-cultured on nutrient agar at 37 °C overnight. To prepare the seed culture, a single colony of each bacterial isolate was cultured in 30 mL of sterilized nutrient broth and incubated overnight at 33 ºC on a shaker at 150 rpm. Subsequently, 0.5 ml of the seed culture was utilized to inoculate 50 ml of nutrient broth, which was incubated for 7 days at 33 °C and 150 rpm. After 7 days, all culture broths were centrifuged for 20 min at 8000 rpm, and then the supernatant of culture broth was used to screen the antibacterial and antifungal activity for each isolate (Balouiri et al. 2016; Ebu et al. 2023).

Evaluation of the antibacterial and antifungal activity

All endophytic isolates were evaluated for antibacterial activity using Muller-Hinton agar medium (MHA) through the agar well diffusion technique as previously described (Balouiri et al. 2016). The bacterial test was performed against the following standard strains, B. subtilis ATCC 6633 and S. aureus ATCC 6538, E. coli ATCC 8739, and P. aeruginosa ATCC 9022. The antifungal activity was evaluated against C. albicans ATCC10231. A fresh culture of the test organisms was adjusted to a count of 0.5 McFarland and was spread by a sterile cotton swab on the surface of the prepared MHA. Then, wells (6 mm) were made in MHA plates using a sterile cork-borer, and 150 µL of supernatant obtained from centrifuged endophyte broth was pipetted to each well individually. The plates were left at room temperature for 15 min to allow diffusion of the supernatant in the agar. The plates were then incubated at 37 °C for 18 h. After incubation, the diameter of the inhibition zone surrounding the wells was measured in mm. The experiment was carried out in triplicate (Sharma and Mallubhotla 2022; Nassar et al. 2023).

Molecular identification of the selected isolates

The selected endophytic isolates were identified using 16 S ribosomal RNA sequence analysis (Clarridge 2004). The PCR products were sequenced at the GATC Biotech (A company using a DNA sequencer (ABI-3730xl) as a partner of Sigma Aldrich, Cairo, Egypt). The resulting sequences were compared with that available in the GenBank with the Basic Local Alignment Search Tool (https://blast.ncbi.nlm.nih.gov/Blast.cgi) to determine the closest related species. The final sequence was aligned using the ClustalX 1.8 software package (http://wwwigbmc.u-strasbg.fr/BioInfo/clustalx). A phylogenetic tree was organized using MEGA (Version 6.1) software based on the neighbor-joining method (Kumar et al. 2018). The confidence level of branching and clustering patterns (1,000 replications) was assessed by bootstrap analysis.

Factors affecting the production of biologically active metabolites

The effects of the incubation time, fermentation medium composition, and environmental parameters such as incubation temperature, media pH, and agitation rate were investigated for maximum production of the bioactive metabolite(s).

Incubation time

A time course experiment was used to evaluate the optimal time for producing antimicrobial metabolites with the greatest growth inhibition against the tested strains. For each isolate, 10 Erlenmeyer flasks (250 mL) having 50 mL of nutrient broth were used. Then, 0.5 ml of the seed culture prepared above was inoculated into the flasks and cultured at 33 ºC for 10 days with 150 rpm agitation and a pH of 7. Each day, one flask was withdrawn from the incubator, centrifuged, and about 150 µL of the supernatant was obtained for evaluating the antimicrobial activity as mentioned above.

Carbon sources

For each bacterial isolate, three Erlenmeyer flasks (250 mL) with 50 mL of the nutrient broth containing two grams of either sucrose, galactose, or starch were used. Then, the respective culture broth was inoculated with 0.5 mL of the seed culture prepared as mentioned above. The flasks were then incubated at 33 ºC for 4 days with an agitation rate of 150 rpm with adjusted initial pH of 7. After incubation, the obtained broths were centrifuged, and 150 µL was used for evaluating the antimicrobial activity as mentioned above.

Nitrogen sources

For each bacterial isolate, sodium nitrate, tryptone, and beef extract at a concentration of 2 g/L were added individually to three Erlenmeyer flasks (250 mL) with 50 mL of the nutrient broth containing the carbon source that gave maximum production of the bioactive metabolites. Each flask was inoculated with 0.5 mL of the seed culture prepared as mentioned before and incubated at 33 ºC for 4 days at 150 rpm and adjusted initial pH of 7. After incubation, the obtained broths were centrifuged, and 150 µL was used to evaluate the antimicrobial activity as mentioned above.

Optimizing environmental factors

The effect of different environmental factors such as temperature (A), pH (B), and agitation rate (C) on metabolite production was optimized using a central composite design (CDD) from response surface methodology. In total, 16 runs were performed for C6 isolate against B. subtilis and 16 runs for C11 isolate against C. albicans, using the optimal carbon and nitrogen sources, and the factors and levels used for these experiments are as follows: temperature: 29, 33, and 37 ºC, pH: 6, 7 and 8 and agitation rate: 150, 200 and 250 rpm. A single response, IZ diameter (mm), was determined after incubation for 4 days. The mean of three measurements was recorded for each run. The design of experiments was carried out by Design Expert® v. 7.0 (Design Expert® Software, Stat-Ease Inc., Statistics Made Easy, Minneapolis, MN, USA). To validate the obtained model, ANOVA (Analysis of variance) was used. Optimum conditions were obtained using the numerical optimization function in the same software (Thompson et al. 1999).

Diagnostic plots

Four plots were built for each isolate to validate our models. A normal probability plot reveals if the residuals stick to a normal distribution. Box-Cox plot on the other hand is used to decide the best power transformation. The predicted vs. actual plot shows how well the model predicts our responses. Finally, Residuals vs. Run plot tests for lurking variables that may have altered the results during the experiments (Design Expert Version 7 User’s Guide).

Evaluation of the antiviral, antioxidant, and cytotoxic activityExtraction of the bioactive metabolites

About 3 mL of the seed culture of the selected isolate was inoculated in 300 mL of nutrient broth containing the optimal carbon, nitrogen sources, and environmentally optimized as described above. The obtained broth cultures were centrifuged at 8000 rpm for 20 min, and the liquid supernatant was mixed with the same volume of ethyl acetate and shaken strongly for 30 min. The mixture was filled in a separating funnel to separate the phase of the organic solvent containing bioactive metabolites. Then, the organic solvent was evaporated by a rotary evaporator at 50 ºC to yield the crude extract. For antiviral, antioxidant, and cytotoxic assays, 1 mL of dimethyl sulfoxide (DMSO) was utilized to dissolve the crude extract (Mamarasulov et al. 2023).

Antioxidant assay

The antioxidant effectiveness of the crude extract of metabolites produced by endophytic bacteria was assessed using the 1, 1- diphenyl-2-picryl hydrazyl (DPPH) free radical scavenging technique. During this procedure, nine concentrations of each sample were prepared as follows, 1000, 500, 250, 125, 62.5, 31.25, 15.62, 7.81, and 3.9 µg/mL. Then about 3 mL of the prepared solution was transferred to a test tube containing one mL of DPPH solution (0.1 mM) and rapidly shaken before being incubated for 30 min at room temperature. Under the same conditions, ascorbic acid was used as a positive control. A spectrophotometer was used to measure the absorbance of the DPPH radical solutions at 517 nm. The DPPH free radical scavenging percent was calculated by the following equation:

$$ }\;}\;}\;\% = \left[ }}/}} \right] \times 00 $$

Ao = absorbance of the control at 517 nm, and A1 = the sample absorbance. The extract’s median scavenging concentration (IC50) of the extract against DPPH was calculated graphically as previously reported (Sulistiyani et al. 2016).

Assay of cytotoxicity

The cytotoxicity of the crude extract of metabolites produced by the respective endophytic isolate was evaluated using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide) (MTT) cell proliferation assay against PC3 (prostatic small cell carcinoma) and Vero cells (Cercopithecus aeithiops kidney epithelial normal cell) according to William and Surin (2014).

In this assay, PC3 and Vero cells were individually inoculated into 96-well tissue culture plates with an intensity of 1 × 105/ mL (100 µL / well) and incubated for 24 h at 37 °C. When the monolayer sheet had fully formed, it was rinsed two times with washing media. Different concentrations of the tested bacterial extracts were prepared by two-fold dilution (1000, 500, 250, 125, 62.5, and 31.25 µg/mL) in RPMI medium with 2% serum (as a maintenance medium). Following that, 0.1 ml of each prepared dilution was added to each well except for three wells (utilized as a control) and received 0.1 mL of the maintenance medium. The plate was then incubated at 37 °C for 24 h. After that, an inverted microscope was used to observe any physical indicators of toxicity such as cell granulation, shrinkage or rounding of the cell, or partial or total loss of monolayer cells. Then, 20 µL of MTT solution (5 mg/mL in phosphate-buffered saline solution, PBS) was added to the wells and shaken thoroughly for 5 minutes at 150 rpm before being incubated at 37 °C with 5% CO2 for four hours. After discarding the MTT solution, each well was filled with 200 µL of DMSO and shaken rapidly for 5 minutes at 150 rpm to suspend the formazan crystal (William and Surin 2014). ELISA reader 76 (Laboratory of Science Way for Scientific Researches and Consultations, AL-Moqattam, Cairo) was used to measure well absorbance (A) at 560 nm. The results were provided as cell viability percentages against untreated control cells. Triplicate wells were assayed, and standard deviations were calculated.

The cell viability and inhibition were measured as shown in Eqs. 1 and 2.

$$ }\;}\;\left( \% \right) = \left( }\;}\;}\;}/}\;}\;}\;}\;}} \right) \times 00 $$

(1)

$$ }\left( 00\% } \right) \, = 00 \, }\% $$

(2)

Antiviral activity assay

The antiviral activity experiment was performed against Herpes Simplex virus type 1 (HSV1). Initially, the maximum non-toxic concentration (MNTC) of the tested extracts on Vero cells was evaluated using the MTT assay. After that, cells with an intensity of 1 × 104 cells/200 µL were introduced to each well in a 96-well plate, except for three wells that were kept for blank controls. The plate was then incubated at 37 °C with 5% CO2 overnight, to allow adherence of cells to the wells. A suspension from an equal volume (1:1 v/v) of the virus and nonlethal dilution of the tested extract was prepared and incubated for one hour. Following that, the wells were filled with 100 µL of viral/sample suspension, agitated at 150 rpm for 5 min, then incubated for 24 h at a temperature of 37 °C with 5% CO2. Cell viability was calculated using the MTT method as explained above.

Genomic analysis for identification of the metabolite biosynthetic gene clustersDNA extraction

The Qiagen DNeasy power- kit (Qiagen, Hilden, Germany) was used to extract DNA in accordance with the manufacturer’s instructions. The Qubit fluorometer ver. 4.0 (Thermo Fisher Scientific, Waltham, Massachusetts, USA) was used to measure the concentration of DNA to confirm that it was at least 55 ng/µL, in accordance with the Oxford Nanopore Standard Operating Procedure (Eltokhy et al. 2021).

Library construction

A total of 34 µL sequencing buffer, 25.5 µL of loading beads, and 4.5 µL nuclease-free water were added and mixed with 12 µL DNA. A Rapid Sequencing Kit (ONT, Oxford, UK) was used to construct the library. Following the fabrication of the library, priming and loading into the FLO-MIN106 (Nanopore Technology, Oxford, England) flow cell was carried out (Eltokhy et al. 2021).

Sequencing and data analysis

The DNA Sequences were performed using the MinION™ (ONT, Oxford, UK). After a full day, 3.03 M reads are produced, and 9.29 K is the N50. Guppy generated real-time base calling during the sequencing process. FAST5 and FASTq files were the format of the output; readings lower than Q7 were not included. Centrifuge software was used to classify the sequences to taxonomy identities (Kim et al. 2016). The Centrifuge index was created using human reference genome (GRCh38), bacterial, and viral genomes that were retrieved from the National Centre of Biotechnology Information (NCBI) RefSeq. Low-complexity areas in the reference sequences with a dust score greater than 20 were masked using the NCBI’s Dust Masker (v1.0.0). The results were visualized using Re-centrifuge (Martí 2019).

Identification of the secondary metabolite gene cluster(s)

The Antibiotics and Secondary Metabolite Analysis Shell (AntiSMASH version 2) was used to align and analyze the sequences for likely secondary metabolite gene clusters (https://antismash.secondarymetabolites.org/#!/start (accessed on 10 June 2024). As previously mentioned, the genome comparison was drafted using the Mauve software (https://gel.ahabs.wisc.edu/mauve) (Eltokhy et al. 2024).

Statistical analysis

All trials were carried out thrice; the error bars indicate the standard deviation. The obtained results were entered and analyzed in the Microsoft Excel program. The SAS version 9.1 software package was used for Analysis of variance (ANOVA) (Thompson et al. 1999). Statistically significant differences were demonstrated by p < 0.05. Design Expert v.7.0 was used for creating the experiment design, model diagnostic plots, response surfaces, and ANOVA analysis.