Antimicrobial and antioxidant activities of Streptomyces species from soils of three different cold sites in the Fez-Meknes region Morocco

Abstract

The increasing demand for new bioactive compounds to combat the evolution of multi-drug resistance (MDR) requires research on microorganisms in different environments in order to identify new potent molecules. In this study, initial screening regarding the antimicrobial activity of 44 Actinomycetes isolates isolated from three soil samples from three different extremely cold sites in Morocco was carried out. Primary and secondary screening were performed against Candida albicans ATCC 60,193, Escherichia coli ATCC 25,922, Staphylococcus aureus ATCC 25,923, Bacillus cereus ATCC 14,579, other clinical MDR bacteria, and thirteen phytopathogenic fungi. Based on the results obtained, 11 active isolates were selected for further study. The 11microbial isolates were identified based on morphological and biochemical characters and their molecular identification was performed using 16S rRNA sequence homology. The UV-visible analysis of dichloromethane extracts of the five Streptomyces sp. Strains that showed high antimicrobial and antioxidant (ABTS 35.8% and DPPH 25.6%) activities revealed the absence of polyene molecules. GC-MS analysis of the dichloromethane extract of E23-4 as the most active strain revealed the presence of 21 volatile compounds including Pyrrolopyrazine (98%) and Benzeneacetic acid (90%). In conclusion, we studied the isolation of new Streptomyces strains to produce new compounds with antimicrobial and antioxidant activities in a cold and microbiologically unexplored region of Morocco. Furthermore, this study has demonstrated a significant (P < 0.0001) positive correlation between total phenolic and flavonoid contents and antioxidant capacity, paving the way for the further characterization of these Streptomyces sp. isolates for their optimal use for anticancer, antioxidant, and antimicrobial purposes.

Figure 1

Primary screening (antibacterial activity) using the double layer method on ISP2 medium.

Figure 2

Antibacerial activity of pure Actinomycetes isolates by the double layer method on ISP2 medium.

Figure 3

Primary screening (antifungal activity) by the double layer method on ISP2 medium against Candida albicans ATCC 60,193.

Figure 4

Primary screening (antifungal activity) by the double layer method on ISP2 medium against phytopathogenic fungi.

Figure 5

Antifungal activity of Actinomycetes pure isolates by the double layer method on ISP2 medium.

Figure 6

Secondary screening (antibacterial activity) by the disc diffusion method on MH medium.

Figure 7

Secondary screening (antifungal activity) by the disc diffusion method on MH medium.

Figure 8

Kinetics of pH evolution and production of antibiotics against Bacillus cereus ATCC 14,579.

Figure 9

Dendrogram showing the evolutionary relationship of the Actinomycetes isolates.

Figure 10

Phylogenetic tree based on 16S rRNA gene sequences of Actinomycetes isolates.The phylogenetic tree based on the 16S rRNA gene was established by statistical method (Neighbour-Joining Tree test), showing the evolutionary relationships among the 11 Actinomycetes isolates marked in red circle and their closest known taxa. Each branch in the tree is associated with a bootstrap value (a percentage ranging from 0 to 100%) which indicates the number of times it has been found during the repetitions and thus determines its credibility, only the values (> 40%) are displayed. The bar (0.010), represents the number of substitutions per nucleotide position (1% of divergence between sequences). GenBank accession numbers are shown in parentheses.

Figure 11

Pearson correlation graphics. (A) Pearson correlation between ABTS and total phenolic content, (B) Pearson correlation between ABTS and flavonoids content, (C) Pearson correlation between DPPH and total phenolic content, and (D) Pearson correlation between DPPH and flavonoids content. “****” (P < 0.0001) highly significant between tests.

Figure 12

UV–visible spectra for the five active crude dichloromethane extracts. (A) Brut extract of Streptomyces sp. E23-2 OM883988, (B) brut extract of Streptomyces sp. E23-4 OM883990, (C) brut extract of Streptomyces sp. E23-11 OM883994, (D) brut extract of Streptomyces sp. E25-11 OM883997, (E) brut extract of Streptomyces bellus E25-12 OM883998.