Synthesis of antibacterial silver nanoparticles from actinomycetes isolated from Thika industrial waste dump sites soil and molecular identification of potential actinomycetes

Abstract

Infectious diseases caused by antibiotic resistant bacteria lead to considerable increase in human morbidity and mortality globally. This requires searching for potential actinomycetes isolates from undiscovered habitats as source of effective bioactive metabolites. The purpose of the present study was to identify actinomycetes isolates from waste dump sites that produce bioactive metabolites capable of synthesizing antibacterial silver nanoparticles. Soil samples were collected from selected waste dump sites and composite soil samples prepared. Composite soil samples were pre-treated with heat and CaCO3. Soil suspension (0.1 ml) from 10-5 serially diluted composite soil sample was spread on selective media for selective growth and isolation of actinomycetes. The primary and secondary screenings of antibacterial active isolates were done by streak plate and well diffusion assay, respectively. The metabolites produced by the KDT32 and KGT32 isolates were harvested and mixed with 10 mM AgNO3 solution for silver nanoparticle synthesis. The synthesis of nanoparticle was confirmed by visual detection and UV-vis spectrophotometer analysis whereas functional groups involved for synthesis was identified by FTIR spectrophotometer. The antibacterial activity of silver nanoparticle was tested in vitro by well diffusion assay. The identification of the potential isolates was done based on 16s rRNA gene sequence analysis using databases from NCBI and EzTaxon-e. The bioassay guided primary screening result showed that from 125 isolates, 29 (23.2%) isolates showed antibacterial activity. From these, isolate KGT22 showed 30+0 mm, 31.3+0.6 mm, 30+0 mm and 36+1 mm inhibition zone against E. coli ATCC 25922, S. boydii, S. typhi and V. cholerea, respectively. Isolate KDO24 showed antibacterial activity against both MRSA (16.25+0.5 mm) and E. coli (26.5+0.58 mm). From 29 isolates, supernatants from 11(37.93%) isolates showed antibacterial activity during secondary screening. Supernatant from BML45, KGT31 and PLS34 showed 17+1 mm inhibition xv zone against E. coli ATCC25922, S. boydii and S. typhi, respectively. The visual detection showed that dark salmon and pale golden rod color change was observed due to the formation of KDT32-AgNP and KGT32-AgNP, respectively. The synthesis was confirmed by a characteristic UV-spectra peak at 415.5 nm for KDT32-AgNP and 416 nm for KGT32-AgNP. The FTIR spectra revealed that OH, C=C and S-S functional groups were involved for the synthesis of KDT32-AgNP whereas OH, C=C and C-H were involved for the formation of KGT32-AgNP. The inhibition zone results revealed that KDT32-AgNP showed 22.0+1.4 mm and 19.0+1.4 mm against E. coli and S. typhi whereas KGT32-AgNP showed 21.5+0.7 mm and 17.0+0.0 mm, respectively. The sequence similarity search result revealed that KDT32 isolate showed 99.8 % sequence similarity with S. lavenduligriseus NRRL ISP-5487 whereas KGT32 isolate showed 99.79 % similarity with S. albidoflavus DSM40455, S. koyangensis strain VK-A60, S. hydrogenans strain NBRC 13475, S. daghestanicus strain NRRL B-5418 and S. violascens strain ISP 5183. The NJ and ML tree revealed that KDT32 isolate formed monophyletic clade with S. nodosus strain ATCC 14899 that showed 99.7% sequence similarity. However, KGT32 formed a distinct clade with S. fabae T66 and S. cinerochromogenes NBRC13822 that showed 99.68 % and 99.58 % sequence similarity, respectively. Isolates produce bioactive metabolites capable of synthesize antibacterial silver nanoparticles, were identified as genus Streptomyces and deposited in GenBank as S. pausti KDT32 (MH301089) and S. pausti KGT32 (MH301090). The synthesis of antibacterial nanoparticle using the bioactive metabolites from such isolates is the first report in this area. Due to its bactericidal activity of the synthesized silver nanoparticles can be used for antibacterial activity in different biomedical applications.