Characterisation of silver nanoparticles synthesised from Serratia sp. strain AQ5-NT39 and its antibacterial potential against pathogenic Vibrios

Marine aquatic bacterial pathogens have plagued the marine aquaculture industry resulting in high mortality rates. Vibrio spp. is one of the strongest marine bacterial pathogens that have become resistant to commercially available antibiotics, thus requiring a potential alternative to combat this pathogenic genus. This present study was done to fulfil the general objectives, which are to isolate marine bacteria with the potential of producing silver nanoparticles (AgNPs) from the marine environment and study their antibacterial potential against the aquatic pathogens Vibrio alginolyticus and Vibrio harveyi. 148 marine bacteria were isolated from marine soil sediments; the isolated bacteria were subjected to screening for the synthesis of AgNPs and tested for antibacterial effects against V. alginolyticus and V. harveyi. The one sample with the best characterisation results and antibacterial effect was identified and further characterised using UV-Visible spectroscopy (UV-Vis), High-resolution transmission electron microscopy (HR-TEM), Energy dispersive X-ray (EDX), Dynamic light scattering (DLS), X-ray diffraction (XRD) and Fourier-transform infrared spectroscopy (FTIR). Meanwhile, optimisation was done using one-factor-at-a-time (OFAT), and response surface methodology (RSM) approaches. The optimised AgNPs were subjected to in vitro and preliminary in vivo antibacterial testing. Serratia sp. strain AQ5_NT39 was identified as the best AgNP sample due to having a sharp peak 440 nm and biggest zone of inhibition. Through the OFAT log phase, 6 h, 0.5 M, 40°C and pH 7 were determined to be the best parameters for the optimal synthesis of AgNPs. RSM determined that interactions between factors were insignificant, while A (p = <0.0001), A2 (p = <0.0001), B2 (p = 0.0002), C2 (p = <0.0001), D2 (p = <0.0001) and E2 (p = 0.0005) representing growth phase, incubation time (h), silver nitrate concentration (M), temperature (°C) and pH were significant. The generated model was determined to be significant with a pvalue of <0.0001. UV-Vis displayed a peak at 420 nm, while HR-TEM determined AgNPs’ size range of 1-100 nm with a non-homogenous mixture of shapes, EDX determined Ag as a primary component, DLS showed a size average of ~96.58 with a PDI average of 0.106, FTIR analysis confirmed the involvement of a protein in the synthesis of AgNPs and XRD analysis determined angles 38.38°, 45.45°, 65.26° and 77.32° with corresponding (hkl) values (111), (200), (220) and (311) planes corresponding with silver planes. In vitro antibacterial testing determined a minimum inhibitory concentration (MIC) of 22.5 ppm and minimum bactericidal concentration (MBC) value of 25 ppm of AgNPs against V. alginolyticus and V. harveyi. Kirby-Bauer disk diffusion at 22.5 ppm of AgNPs demonstrated an average zone of inhibition of 19.0 mm and 18.3 mm against V. alginolyticus and V. harveyi, respectively. The growth inhibition curve displayed that the concentration of V. alginolyticus and V. harveyi was 106 CFU mL-1. The preliminary in vivo analysis showed an increase in the survival rate of Artemia nauplii challenged with V. alginolyticus and V. harveyi pre-treated with optimised AgNPs sample AQ5-NT39 of 33.33% and 31.67%, respectively as compared to untreated samples, whereas the Vibrio loads in the Artemia (V. alginolyticus: Log10 5.81 ± 0.115 and V. harveyi: Log10 6.59 ± 0.067) and culture water (V. alginolyticus: Log10 6.03 ± 0.095 and V. harveyi: Log10 6.72 ± 0.011) were significantly reduced after treatment. This research has provided sufficient data for the potential use of AgNPs as an alternative to commercial antibiotics in the marine aquaculture industry in Malaysia.

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