Broad Spectrum Antimicrobial Activity of Forest-Derived Soil Actinomycete, Nocardia sp. PB-52

Abstract

A mesophilic actinomycete strain designated as PB-52 was isolated from soil samples of Pobitora Wildlife Sanctuary of Assam, India. Based on phenotypic and molecular characteristics, the strain was identified as Nocardia sp. which shares 99.7% sequence similarity with Nocardia niigatensis IFM 0330 (NR_112195). The strain is a Gram-positive filamentous bacterium with rugose spore surface which exhibited a wide range of antimicrobial activity against Gram-positive bacteria including methicillin-resistant Staphylococcus aureus (MRSA), Gram-negative bacteria, and yeasts. Optimization for the growth and antimicrobial activity of the strain PB-52 was carried out in batch culture under shaking condition. The optimum growth and antimicrobial potential of the strain were recorded in GLM medium at 28°C, initial pH 7.4 of the medium and incubation period of 8 days. Based on polyketide synthases (PKS) and non-ribosomal peptide synthetases (NRPS) gene-targeted PCR amplification, the occurrence of both of these biosynthetic pathways was detected which might be involved in the production of antimicrobial compounds in PB-52. Extract of the fermented broth culture of PB-52 was prepared with organic solvent extraction method using ethyl acetate. The ethyl acetate extract of PB-52 (EA-PB-52) showed lowest minimum inhibitory concentration (MIC) against S. aureus MTCC 96 (0.975 μg/mL) whereas highest was recorded against Klebsiella pneumoniae ATCC 13883 (62.5 μg/mL). Scanning electron microscopy (SEM) revealed that treatment of the test microorganisms with EA-PB-52 destroyed the targeted cells with prominent loss of cell shape and integrity. In order to determine the constituents responsible for its antimicrobial activity, EA-PB-52 was subjected to chemical analysis using gas chromatography-mass spectrometry (GC-MS). GC-MS analysis showed the presence of twelve different chemical constituents in the extract, some of which are reported to possess diverse biological activity. These results confirmed that the presence of bioactive constituents in EA-PB-52 could be a promising source for the development of potent antimicrobial agents effective against wide range of microbial pathogens including MRSA.

Bioactive potential of actinobacteria isolated from certain under-studied regions in India

“…Terrestrial samples yielded more colonies on starch casein agar whereas marine sediments yielded more colonies on Kusters agar. Many authors reported Streptomyces as a major actinobacterial population in both terrestrial (Radhakrishnan et al, 2007;Mohanraj et al, 2014) and marine ecosystems (Sivakumar et al, 2005;. In the present study also majority of the recovered actinobacterial cultures are Streptomyces (86%).…”

“…Further the heat treatment method and antibacterial and antifungal antibiotic supplemented in the isolation medium resulted in the reduction of unwanted bacterial and fungal growth, respectively. Cross streak method using modified nutrient glucose agar (MNGA) is the most common method used for the detection of antagonistic actinobacteria (Radhakrishnan et al, 2007;Balagurunathan et al, 2001).…”

“…Leaves sample from the mangrove plant Rhizophora apiculata was also collected from Parangipettai coastal area, Tamil Nadu (Table 1). All the soil and sediment samples were air-dried at room temperature for 3-5 days and the sieved samples were kept at 55°C for 10 minutes in a glass container (Radhakrishnan et al, 2007). Plant leaves were pretreated by adopting three step process described by Coombs and Franco (2003).…”

“…The same procedure was followed for all the remaining samples. All the plates were incubated at 28°C for one month (Radhakrishnan et al, 2007).…”

“…However, due to overuse or misuse of antibiotics over a prolonged period, most of the pathogens have become resistant to the antibiotic therapy. Thus, there is a dire need for the discovery and development of new antibiotics to effectively target the life threatening disease causing pathogens (Sharma et al, 2016). Microbial resources are reported as promising source for novel metabolites.…”