Preparative Screening of Desert Actionbacteria active against Methicillin Resistant Staphylococcus aureus (MRSA)

Abstract

This study aimed at the exploration of a harsh ecological niche ―the Cholistan desert Pakistan‖ for actinobacterial diversity, and to investigate their potential for the production of inhibitory compounds against methicillin resistant Staphylococcus aureus (MRSA). The 20 sand and soil samples were collected from various locations of Cholistan desert located in southern Punjab, Pakistan. A total of 110 actinobacterial strains were isolated and were tested for antimicrobial activity, among them 30 strains showed inhibitory effects against MRSA, which were selected for further studies. The taxonomic characterization of these strains strongly suggested that they were the members of different families of the order Actinomycetales. The most frequently isolated strains belonged to the family Streptomycetaceae, and 29 different species of genus Streptomyces were identified, while one strain belonged to the family Pseudonocardiaceae. The MRSA strains used in this study, were obtained from a clinical laboratory and were identified and validated as MRSA by different morphological, biochemical and genetic approaches. The validation of these strains as MRSA was done using cefoxitin disc susceptibility testing according to the CLSI standards and by the PCR amplification and sequencing of 16S rRNA and mec-A genes. The susceptibility pattern of all these strains against different antibiotic classes was also determined and these were found to be resistant to penicillin and its derivatives (amoxicillin, ampicillin and piperacillin) and also against the cephalosporins (cefpirome, cephalexin, cefoperazone). The MRSA strains were also tested for the vancomycin susceptibility and were found to be sensitive. xiv For screening purpose, the selected actinobacterial strains were grown as shaking flask cultures on lab scale and the resulting methanolic extracts were screened biologically and chemically. In biological screening, the actinobacterial strains exhibited significant antiMRSA activity in the range of 20-32mm inhibitory zones. In chemical profiling by thin layer chromatography, the methanolic extracts revealed the capability of the strains to produce a variety of secondary metabolites, as indicated by the appearance of colored bands on TLC after staining with anisaldehyde/H2SO4. The HPLC/UV and LC-MS analysis also provided a thorough understanding of the chemical and metabolic capacity of the selected strains in the form of various peaks at different retention times (tR). On the basis of anti-MRSA activity and significant chemical profile, the strains were prioritized for preparative screening for the purification and identification of the active compounds and for genomic studies. Among the prioritized strains, one of the strains (AFD22) yielded 4 new and 3 known compounds (Actinomycin D, Actinomycin X0β and N1S-(4-methylaminophenylmethyl)-2-oxo-propyl acetamide) which were identified by MS and NMR analysis. Similarly, the other prioritized strains (AFD18 and AFD26) were also found to produce bioactive compounds which were partially identified by LC-MS analysis and by comparison of the data in a database (AntiBase). One of the prioritized strains (AFD10) was investigated by a sort of indirect approach using NGS whole genome sequencing and genome mining techniques and biosynthetic gene clusters encoding different antibiotics and secondary metabolites were detected by antiSMASH analysis, which were further confirmed by the LC-MS profiling of the methanolic extracts from three different media. Overall, the study revealed that the Cholistan desert Pakistan harbors an immense diversity of actinobacteria and most of the strains produce structurally diverse bioactive secondary metabolites, which are a promising source of novel drug candidates against MRSA.