Discovery of Novel Antibacterial Agents Targeting MRSA Pathogens from Unexploited Actinomycetes

Abstract

Background: Antimicrobial resistance (AMR) has been present ever since the discovery of the first classes of antibiotics, where Sir Alexander Fleming warned about it in his Nobel Prize speech back in 1945. With the lack of new antibiotic classes and the continuous emergence of resistant bacteria, it is estimated that AMR will be killing 10 million people yearly by the year 2050 if no significant measures are taken. Discovering new antibacterial classes in addition to unexploited targets should be the main goal to overcome this problem. Natural resources, such as the soil, harbor microorganisms, Actinomycetes that can produce secondary metabolites that could have antibacterial activity, and could, therefore, be used in the search for new antibacterial classes. According to the World Health Organization (WHO) pathogen priority list, Staphylococcus aureus Methicillin-resistant (MRSA) was considered as a high priority pathogen in 2017 which makes it a relevant target for these prospective novel agents. This study aims to purify novel Actinomycetes from soil samples and screen them against a panel of pathogenic bacteria to discover new classes and targets of antibacterial agents.

Methods: Soil samples were collected from different regions in Lebanon and were dried, heated, and serially diluted before streaking on ISP3 and soil agar. The observed bacterial colonies were purified based on their morphology and stored in glycerol. The bacteria were then put under stress in 14 different production media and the secondary metabolites produced were extracted using Amberlite XAD-16N resin, methanol, and acetone and screened for antibacterial activity on Staphylococcus aureus ATCC 29213, Staphylococcus aureus Newman, Staphylococcus aureus N315, Enterococcus faecalis ATCC 19433, Klebsiella pneumoniae DSM, Acinetobacter baumannii DSM 30008, Klebsiella pneumoniae ATCC 13883, Pseudomonas aeruginosa MEXAB, and Escherichia coli ATCC 25922 using a broth microdilution assay. The medium that showed the highest number of wells with inhibition was then up-scaled and further separated into fractions. DNA was also extracted from bacteria of interest and 16S rRNA sequencing in addition to whole-genome sequencing was performed to identify novel Actinomycetes strains. NaCl and pH tolerance tests were also performed on the strain.

Results: The extract produced by strain ZK3 showed significant inhibition in medium RA3 on Gram-positive bacteria including Staphylococcus aureus N315, and Enterococcus faecalis ATCC 19433. Inhibition was also evident in the ethyl acetate and the chloroform fractions after separation of the upscale. The sequenced PCR product of 16S rRNA was blasted and the results show that the strain belongs to the Streptomyces species. Significant biosynthetic gene clusters were evident in the antiSMASH results of the WGS product. Testing also showed that 0% NaCl concentration and pH 7 were optimal conditions for growth.

Conclusion: In this study, we were able to isolate bacteria from soil, harboring inhibitory activity against MRSA, and other Gram-positive bacteria. The extract produced by this bacteria should be further purified and tested to discover the nature and the structure of the compound in addition to its mode of action.