This Is AuburnElectronic Theses and Dissertations

Discovery and characterization of antibacterial compounds expressed by soil microorganisms using culture-dependent and -independent approaches




Nasrin, Shamima

Type of Degree



Biological Sciences


The emergence of multidrug-resistant pathogens has increased the demand for discovery of novel antibiotics. Both culture-dependent and culture-independent approaches were used to discover antibiotics against methicillin-resistant clinical isolates of Staphylococcus aureus (MRSA). A collection of 548 bacterial and fungal isolates were isolated from soil using low-strength (1/200th) nutrient agar supplemented with soil extract incubated for more than three months. Two isolates, designated as A115 and F4, were found to inhibit the growth of pathogenic MRSA strains. The isolate A115, member of the genus Streptomyces, produces pink pigments after extended incubation. The isolate F4, identified as Nonomuraea, produces a high molecular weight (>100kDa), heat-stable reddish pigment with anti-MRSA activity. Whole genome sequencing using a combination of shotgun and mate-pair next-generation sequencing resulted in the complete assembled genome for each isolate, with the size of the A115 and F4 genomes at 8.6 Mbp and 10.3 Mbp, respectively. The %G+C contents of strains A115 and F4 were determined to be 71% and 70.4%, respectively. Phylogenetic analysis using six housekeeping genes revealed that strain A115 was most closely related to Streptomyces afghaniensis and Streptomyces olindensis; however, the low level of average nucleotide identity (ANI) values in comparing the A115 genome were 89.76% and 89.14% for S. afghaniensis and S. olindensis, respectively. These genomic results, combined with differentiation of strain A115 from other Streptomyces species by morphological and physiological characteristics, led to the conclusion that strain A115 is a novel species of the genus Streptomyces, for which the name Streptomyces alburnustigris sp. nov. is proposed. In silico analysis using anti-SMASH predicts that A115 and F4 genomes encode many genetic clusters for secondary metabolite biosynthesis, including the synthesis of terpene, aminoglycoside, thiopeptide, bacteriocin, oligosaccharide, phenazine, butyrolactone, siderophore, melanine and potentially other bioactive compounds produced by non-ribosomal peptide synthetase and polyketide synthetase pathways. Both the genomes of S. alburnustigris A115 and Nonomuraea spp. strain F4 are predicted to encode Type I, II, and III PKS pathways. A large collection of plant growth-promoting rhizobacteria (PGPR) (n=147) isolates were screened for anti-MRSA activity, among which five Bacillus strains were identified with anti-MRSA activity. One of these five, B. amyloliquefaciens strain AP183, was found to produce a novel macrodiolide compound described herein as bacillusin A with potent anti-MRSA activity of a minimum inhibitory concentration of 0.6 µg/mL. Based on itsnovel biochemistry and strong in vivo anti-MRSA activity, strain AP183 was selected for evaluation as a skin probiotic to prevent MRSA infection using a mouse wound model. In vivo studies showed that co-administration of secondary metabolites and AP183 spores resulted in a significant reduction in the number of S. aureus that colonized mouse skin compared to a negative control. Analysis of 16S rRNA genes PCR amplified from skin samples revealed a significant reduction in the relative abundance of S. aureus after AP183 application while the relative abundance of other bacterial taxa increased in the skin microbiome as a result of probiotic administration. Using a culture-independent approach to identify antibacterial compounds, a large-insert soil metagenomic library was constructed that comprised of 19,200 E. coli clones with an average insert size of 110 kb. The library clones were screened for anti-MRSA activity using a 96-well microtiter plate. In situ lysis of the E. coli host enabled detection of both intra- and extracellular compounds, yielding a total of 28 clones that consistently inhibited MRSA growth. Transformation of naïve E. coli with BAC DNA isolated from anti-MRSA clones confirmed the presence of their anti-MRSA activity. Seven of the clones were capable of modifying chloramphenicol added to the E. coli culture medium, thereby resulting in modification of an existing antimicrobial scaffold. LC-MS analysis of the organic extract of the clones revealed three new chloramphenicol derivatives. Chemical synthesis of these derivatives showed antimicrobial activities against diverse group of pathogens including MRSA, Mycobacterium intracellulare and M. tuberculosis. Together with all these results demonstrate that both culture-dependent and –independent approaches can be used to identify previously undescribed bioactive compounds with antimicrobial activity that can be used to control multidrug-resistant pathogens.