Bacterial strains

All the bacterial strains involved in this study are brought from MIRCEN (Ain Shames University, Egypt): Staphylococcus aureus EMCC 1351, Escherichia coli ATCC 25922, Klebsiella pneumoniae EMCC 1637, Lactobacillus acidophilus EMCC 1324 and Proteus vulgaris ATCC 8427.

Preparation of L. acidophilus filtrate and silver nitrate reduction

The reduction of silver nitrate yielded bionanosilver with Lactobacillus acidophilus filtrate according to the method of Rajesh et al. (2015) as Silver nanoparticles were made using deionized water as a solvent. L. acidophilus starter culture was incubated in Man-Rogosa-Sharpe broth overnight at 37 °C for 48 h. Following incubation, the culture was centrifuged for 30 min at 10,000 rpm to separate the supernatant using Whatmann filter paper no. 1. After that, different ratios of 2:8, 3:7, and 4:6 of culture filtrate to silver nitrate (Sigma, Aldrich) solution were applied to the culture filtrate. The synthesis was then continued for 24 h at 35 °C in the dark. Silver nanoparticle isolation and purification Centrifugation was used to separate the silver nanoparticles and it took 20 min at 10,000 rpm. Two times, sterile distilled water was used to wash the pelleted silver nanoparticles.

Spectral study of UV–visible light

A UV–visible spectrophotometer set to 420 nm was used to track the biosynthesis of AgNPs on a regular basis; 0.1 mL of the sample was obtained and diluted to 2 mL with deionized water for the analysis (El-Aassar et al. 2020).

Pathogenic bacteria

Luria–Bertani (LB) broth media (Becton-Dickinson and Co., USA) were used to culture the bacteria (P. vulgaris, E. coli, S. aureus, and K. pneumoniae). One-mL aliquots of a 24-h-old broth culture of the tested microorganisms were aseptically disseminated onto nutrient agar slopes and incubated for 24 h at 37 °C. The bacterial growth was extracted and washed away with sterile normal saline, yielding a solution with roughly 108–109 CFU/mL. The suspension was kept refrigerated at 4 °C until it was used (Al-Askar et al. 2013).

Cytoxicological effects of bionanosilver on experimental animals

Nine rats about 200 g in weight were divided into three groups to study nanoparticle toxicity in vivo. The three groups were G1, intragastric application with 200 mg/kg AgNPs, sacrificed after 1 week; G2, animals given the same dose but kept for 2 weeks; and G3, the control animals (Lala et al. 2022).

Antibacterial activity of AgNPs

The antibacterial efficacy of the generated AgNPs against harmful bacteria was investigated according to the method of Al-Askar et al. (2013). In addition, biogenic antibiotics (200 µg/mL) such as ampicillin, gentamicin, and streptomycin were used to test the nanoparticles’ bactericidal activity.

Bionanosilver characterization using scanning electron microscopy

One drop of the created AgNPs aqueous suspension was put on a glass slide, and the films on the SEM grids were allowed to dry for 2 min. SEM analysis was carried out on a Hitachi-JP/H7600 apparatus (Japan) with a 100 kV accelerating voltage. With the use of SigmaScan Pro software, the size of the resulting AgNPs was determined based on SEM micrographs (SPSS Inc., Version 4.01.003). Energy dispersive X-ray studies were performed on a JEOL JSM-6400 microscope (Japan) (El-Aassar et al. 2020).

X-ray diffraction and fourier-transform-infrared spectroscopy

X-ray power diffraction (XRD) (Shimadzu XRD 7000 X-ray diffractometer, Japan) was used to explore the structure of the copolymer as well as the formation of AgNPs, and the structure of the AgNPs was evaluated using Fourier-transform-infrared (FTIR) spectra. To make pellets, powder samples were combined with potassium bromide. A Bruker TENSOR Series FTIR spectrometer (Germany) connected to a PC was used to record FTIR spectra in the absorbance mode, and the data were analyzed using IR Solution OPUS™ software (El-Aassar et al. 2020).

Analyzing particle size

A submicron particle size analyzer was used to examine the particle size of AgNPs during their formation (Beckman Coulter, USA). The sample was dispersed in water at 20 °C with a viscosity of 1.002 and a refractive index of 1.33 (El-Aassar et al. 2020).

Total RNA extraction and cDNA synthesis from treated and untreated bacteria

An RNeasy Mini Kit was used to isolate bacterial RNA from both treated and untreated bacteria, according to the manufacturer’s recommendations (Qiagen, Germany). The isolated RNA was treated with DNase for 1 h at 37 °C to eliminate any DNA residue. Reverse transcription of purified total RNA into cDNA was performed in a reaction volume of 25 µL. Included in the reaction mixture were 2.5 µL of 5× buffer with MgCl2, 2.5 µL of 2.5 mM dNTPs, 4 µL of oligo (dT) primer (20 pml/L), 2 g RNA, and 200 U reverse transcriptase enzyme (MLV, Fermentas, USA). A thermal cycler (Eppendorf, Germany) was used to perform RT-PCR amplification at 42 °C for 1 h and 72 °C for 10 min. The cDNA was then kept at − 20 °C until it was needed (El-Aassar et al. 2020).

PCR with differential display

The reverse primers of the mecA and beta-lactam genes were used to trace the mRNA of the two genes in the bacteria treated with biosynthesized nanosilver. For mecA, R: 5′-AGTGCAGTA CCGGATTTGC-3′ (Kumurya 2015). For beta-lactam, R: 5′-GAGCTCGGTATTGTAATATGATCCTCTAGA-3′ (Jiang et al. 2020). A total amount of 25 µL of the droplet-digital PCR (DD-PCR) reaction mixture was used according to Ramadan (2015). The PCR amplification was carried out in an Eppendorf thermal cycler, which was designed for one cycle at 95 °C for 5 min, followed by 34 cycles as follows: Denaturation at 30 s at 95 °C, annealing 1 min at 45–48 °C, and elongation at 1 min at 72 °C. As a last extension, the reaction was incubated at 72 °C for 10 min. Prior to loading 10 µL per gel slot, 2 µL of loading dye was applied. In 1.5% agarose/0.5× TBE gels, electrophoresis was done at 80 v using 0.5× TBE as running buffer, and the gel was subsequently stained in 0.5 µg/mL (w/v) ethidium bromide solutions and destained in deionized water. Finally, a gel documentation system was used to visualize and photograph the gel. The STATISTICA5 tool was used to evaluate the data based on the up-down-regulated genes.

Sequencing of selected bands and DNA sequence analysis

Four distinct bands (three down-regulated and one uncontrolled) were chosen and purified from the gel using a PCR purification kit (Qiagen, USA), and the purified DNA was sent to be sequenced (Macrogene, Korea). The sequence analysis was performed using the CLUSTALW program (https://www.genome.jp/tools-bin/clustalw) and the phylogeny was constructed using the MEGA 5 program (https://www.megasoftware.net/).

Analytical statistics

Collected data were processed then reported as mean plus or minus standard deviation (mean ± S.D.). Using Excel, one-way-ANOVA (Microsoft Office 2007) was performed to assess the effect of prepared AgNPs on various physiological parameters. A value of P < 0.05 was considered to be statistically significant.