S. Clavifer Produces Antibiotics Against Beta-Lactam Resistant S. Aureus and E. Coli

The number of patients associated with bloodstream infections from methicillin resistant Staphylococcus aureus and Escherichia coli resulted with over 8,000 deaths in 2007. The emergence of antibiotic resistance bacteria is a series and global concern because they are resistant to many commonly used antibiotics. IDSA acknowledged the importance of antibiotic discovery by launching a 10’ x 20’ initiative for the development of 10 new, safe and effective antibiotics by 2020. Actinobacteria, especially Streptomyces sp., are a group of Gram-positive bacteria that are a major source for antibiotic producers. They are known to produce clinically important antibiotics including aminoglycosides, macrolides, and tetracyclines. Streptomyces clavifer have never been reported as antibiotic producers against beta-lactam resistant S. aureus and E. coli. The objectives of our research were (i) to analyze S. clavifer’s antibiotics that are active against beta-lactam resistant S. aureus and E. coli and (ii) to purify antibiotics produced by S. clavifer. S. clavifer was identified using 16S rRNA gene analysis. The bacteria were grown on ISP2 medium for seven days at 26°C. Antibiotics from S. clavifer were extracted twice using 500 mL of ethyl acetate (EtOAc). The extracts were tested for antibiotic activity against methicillin sensitive S. aureus ATCC 25923 (MSSA) and methicillin resistant S. aureus ATCC 43300 (MRSA). S. clavifer was further tested against susceptible and beta-lactam resistant E. coli ATCC 25922 and 35218, respectively. The optimized solvent system and R_f was obtained by thin-layer chromatography (TLC) using different ratios of dichloromethane-methanol (DCM:MeOH). The antibiotics from S. clavifer extract were purified using flash chromatography. The fractions were further tested for purity using RP-HPLC. The relative zone of inhibitions from S. clavifer against S. aureus ATCC 25923 and 43300, and E. coli ATCC 25922 and 35218 were 6.5, 8.5, 5.5 and 5.0 mm, respectively. The extraction of S. clavifer formed a yellow solution and yielded 24.41 mg of brown extracts from 500 mL of culture. The TLC analysis revealed that the optimized solvent system was 95 DCM: 05 MeOH. S. clavifer produced three antibiotics that were effective against both S. aureus ATCC 25923 and 43300 with R_f values 0.74, 0.31 and 0.27. Additionally, S. clavifer produced two antibiotics that were effective against both E. coli ATCC 25922 and 35218 with R_f value 0.74 and 0.31. The antibiotic (R_f  = 0.31) from S. clavifer that inhibited both S. aureus ATCC 43300 and E. coli ATCC 35218 was successfully purified. RP-HPLC-chromatogram showed one dominant peak with retention time of 3.542 minutes suggesting high purity of the sample. Moreover, we discovered that S. clavifer’s antibiotics were stable at temperatures of at least 60^oC. Purifying the antibiotics responsible for the activity against beta-lactam resistant S. aureus and E.coli, as well as identifying the chemical structure of the antibiotics are essential for future investigations.