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.

Keywords: GC-MS; Nocardia sp.; SEM; antimicrobial activity; biosynthetic genes; culturing conditions; microbial pathogens.

Figure 1

Microscopic view showing spore chain of Nocardia sp. PB-52 on GLM agar (A) Micro morphology using cover slip insertion method; (B) Scanning electron micrograph view.

Figure 2

Phylogenetic tree of Nocardia sp. PB-52 and the closest Nocardia species showing phylogenetic relationships based on the 16S rRNA gene sequences using neighbor-joining method. Bootstrap percentages based on 1000 resamplings are listed at nodes, only values above 50% are given. Bar, 0.005 substitutions per nucleotide position.

Figure 3

Effect of different culture media on growth and antimicrobial activity assessed in terms of diameter of inhibition zone by Nocardia sp. PB-52 (Test organism: S. aureus MTCC 96).

Figure 4

In-vitro antimicrobial activity of EA-PB-52 following disc diffusion method against (A) S. aureus MTCC 96 (B) C. albicans MTCC 227 (i) 10% DMSO (negative control), (ii) 20 μg/disc rifampicin (positive control), (iii) 30 μg/disc amphotericin B (positive control), (iv) 20 μg/disc EA-PB-52.

Figure 5

Effect of temperature on growth and antimicrobial activity assessed in terms of diameter of inhibition zone by Nocardia sp. PB-52 (Test organism: S. aureus MTCC 96).

Figure 6

Effect of pH on growth and antimicrobial activity assessed in terms of diameter of inhibition zone by Nocardia sp. PB-52 (Test organism: S. aureus MTCC 96).

Figure 7

Effect of incubation period on growth and antimicrobial activity assessed in terms of diameter of inhibition zone by Nocardia sp. PB-52 (Test organism: S. aureus MTCC 96).

Figure 8

Agarose gel electrophoresis of PCR amplified products of Nocardia sp. PB-52. (A) Selective amplification of PKS-I using K1F/M6R specific primers; (B) Selective amplification of NRPS using A3F/A7R specific primers.

Figure 9

Scanning electron micrograph showing the effect of 1 × MIC EA-PB-52 against P. aeruginosa MTCC 741 (A) without treatment, (B) treatment with EA-PB-52; and against C. albicans MTCC 227 (C) without treatment, (D) treatment with EA-PB-52.

Figure 10

Chemical structures of the identified compounds from EA-PB-52.