Ecofriendly Synthesis of Silver Nanoparticles by Terrabacter humi sp. nov. and Their Antibacterial Application against Antibiotic-Resistant Pathogens

Abstract

It is essential to develop and discover alternative eco-friendly antibacterial agents due to the emergence of multi-drug-resistant microorganisms. In this study, we isolated and characterized a novel bacterium named Terrabacter humi MAHUQ-38^T, utilized for the eco-friendly synthesis of silver nanoparticles (AgNPs) and the synthesized AgNPs were used to control multi-drug-resistant microorganisms. The novel strain was Gram stain positive, strictly aerobic, milky white colored, rod shaped and non-motile. The optimal growth temperature, pH and NaCl concentration were 30 °C, 6.5 and 0%, respectively. Based on 16S rRNA gene sequence, strain MAHUQ-38^T belongs to the genus Terrabacter and is most closely related to several Terrabacter type strains (98.2%-98.8%). Terrabacter humi MAHUQ-38^T had a genome of 5,156,829 bp long (19 contigs) with 4555 protein-coding genes, 48 tRNA and 5 rRNA genes. The culture supernatant of strain MAHUQ-38^T was used for the eco-friendly and facile synthesis of AgNPs. The transmission electron microscopy (TEM) image showed the spherical shape of AgNPs with a size of 6 to 24 nm, and the Fourier transform infrared (FTIR) analysis revealed the functional groups responsible for the synthesis of AgNPs. The synthesized AgNPs exhibited strong anti-bacterial activity against multi-drug-resistant pathogens, Escherichia coli and Pseudomonas aeruginosa. Minimal inhibitory/bactericidal concentrations against E. coli and P. aeruginosa were 6.25/50 and 12.5/50 μg/mL, respectively. The AgNPs altered the cell morphology and damaged the cell membrane of pathogens. This study encourages the use of Terrabacter humi for the ecofriendly synthesis of AgNPs to control multi-drug-resistant microorganisms.

Keywords: AgNPs; Terrabacter humi MAHUQ-38T; eco-friendly synthesis; multi-drug-resistant microorganisms.

Figure 1

Transmission electron micrograph of cells of Terrabacter humi MAHUQ-38^T after negative staining with uranyl acetate, Bar, 1.0 μm.

Figure 2

Neighbor-joining (NJ) tree based on 16S rRNA gene sequence analysis showing phylogenetic relationships of strain MAHUQ-38^T and members of genus Terrabacter. Bootstrap values more than 70% based on 1000 replications are shown at branching points. Scale bar, 0.05 substitutions per nucleotide position.

Figure 3

Reasoner’s 2A (R2A) broth with AgNO3 as a control (A); ecofriendly synthesized silver nanoparticles (AgNPs) (B); ultraviolet–visible (UV–vis) spectra (C); and field emission-transmission electron microscopy (FE-TEM) images of ecofriendly synthesized silver nanoparticles (D,E).

Figure 4

Energy dispersive X-ray (EDX) spectrum of ecofriendly synthesized AgNPs (A); TEM image (electron micrograph region) used for mapping (B); and the distribution of silver in elemental mapping (C).

Figure 5

X-ray diffraction pattern (A) and selected area diffraction (SAED) pattern (B) of ecofriendly synthesized AgNPs.

Figure 6

Fourier transform infrared (FT-IR) spectra of ecofriendly synthesized silver nanoparticles.

Figure 7

Zones of inhibition of ecofriendly synthesized AgNPs (30 μL) at 500 and 1000 ppm concentrations in water against E. coli and P. aeruginosa.

Figure 8

Zones of inhibition of commercial antibiotics against E. coli and P. aeruginosa. Abbreviation: VA (vancomycin, 30 μg/disc), E (erythromycin, 15 μg/disc) and P (penicillin, G 10 μg/disc).

Figure 9

Growth curves of E. coli (A) and P. aeruginosa (B) cultured in Mueller–Hinton broth (MHB) with different concentrations of the synthesized AgNPs to determine the Minimal Inhibitory Concentration (MIC).

Figure 10

MBC of synthesized AgNPs against E. coli (A) and P. aeruginosa (B).

Figure 11

SEM images of normal E. coli cells (A), 1 × MBC AgNPs treated E. coli cells (B), normal P. aeruginosa cells cells (C), and 1 × MBC AgNPs treated P. aeruginosa cells cells (D).