Research Projects

Antimicrobial resistance (AMR), including ESKAPE pathogens, caused 1.27 million deaths and was linked to 4.95 million globally, surpassing HIV/AIDS and malaria fatalities (1). Antimicrobial peptides (AMPs) show promising alternatives to combat resistance(2). In this study, the antimicrobial properties of epiphyte, Pseudomonas sp. UV AK001 (P. Sp. UV AK001), isolated from the bark of a plant in the northern mountains of Kerala, were evaluated. Crude ethyl acetate extracts from the strain P. Sp. UV AK001 demonstrated potent activity against both Gram-negative and Gram-positive bacteria, along with antifungal and anticancer properties. The 16S rRNA sequence was submitted to the NCBI with accession number PP93872, it revealed a 99.79% sequence similarity with P. kribbensis and P. allokribbensis. Indicating a close evolutionary relationship. This study centers on AMPs and their effectiveness against ESKAPE pathogens. In the preliminary process development for the AMPs, optimizing glucose (5 mM) in LB medium for 12 hours of growth (OD 1.6–1.8) at 30°C enhanced antimicrobial activity. Bioactivity declined after 12 hours might be due to proteolytic degradation or feedback inhibition need to be elucidated Conversely, the addition of proline (0.2 mM) in LB medium reduced the production of AMPs. Filtered supernatant (0.45 micron) from A. baumannii and S. aureus exhibited an antagonistic effect, further improving the AMPs production. In solvent extraction optimization, ethyl acetate extracts from the optimized culture demonstrated potent inhibition against all ESKAPE pathogens, surpassing the effectiveness of DCM and hexane extracts. Thin-layer chromatography (TLC) autogram analysis along with ninhydrin test revealed three antimicrobial peptides with retention factor (Rf) values of 0.27, 0.3, and 0.58 AMPs and two AMCs with Rf values of 0.5 and 0.86. Both AMPs and AMCs displayed broad-spectrum activity in well diffusion assays. To explore the antimicrobial potential of AK001, the genome of P. allokribbensis (3) was analyzed using AntiSMASH (4), Four out of 16 biosynthetic gene clusters (BCGs) clusters were novel, with no similarity to known clusters and classified as RiPP-like gene clusters. This study aims to purify and characterize novel AMPs and AMCs from the P. Sp. UV AK001 strain. Genome sequencing will identify novel biosynthetic gene clusters, while RNA sequencing at 6, 12, and 18 hours will analyze the transcriptomic profile during AMPs synthesis in response to glucose, revealing feedback mechanisms. Metabolic profiling will provide insights into peptides and other bioactive compounds. Further, Strain-engineering approaches will be used to overcome feedback inhibition and enhance AMPs production. Additionally, recombinant expression of RiPP-like gene clusters with fusion peptide tags will be explored to enhace peptide yield, paving the way for an industrially scalable P. Sp. UV AK001 variant to combat multidrug-resistant pathogens.