News & Events
Posted on October 21, 2022
Date - October 21, 2022
2:15 am - 3:15 am
Public PhD Defense
Department of Chemistry & Biochemistry
Cech Research Laboratory
Title: New Leads Against Drug-Resistant Organisms: Shifting the Focus in Discovery Towards Rapid Screening and Mechanism of Action
Over the past century, antibiotic therapy has been successful in nearly eliminating a former leading cause of death. Mortality records from the past 100 years in the US show a decrease from 46% of deaths due to infectious diseases, to just 3% in 2010. While the use of antibiotics has had a tremendously positive impact on healthcare, the subsequent rise of antibiotic resistance in pathogens has threatened to reverse recent advances in public health and the study of antibiotic mechanisms has gained significant interest. Issues leading to emergence of antibiotic-resistant pathogens include overprescribing of antibiotics and abuses by the agricultural industry, with 50% of manufactured antibiotics used in agriculture, rather than the medical field as originally intended. Misuse, by failure to regard antibiotics as an inestimable resource, coupled with stagnation in discovery of new antibiotics, has led to the prevalence of antibiotic-resistant pathogens. Approaches to combat drug resistant pathogens include large scale screening of natural sources, such as plants and fungi, for antimicrobial compounds with diverse chemical scaffolds and prioritization of antimicrobials operating by mechanisms of action which bacteria encounter infrequently.
Due to the emergence of resistance, the World Health Organization considers Gram-negative pathogen Acinetobacter baumannii a top priority for therapeutic development. Using this priority pathogen and a phenotypic, agar plate-based assay, a unique library of extracts from 2,500 diverse fungi was screened for antimicrobial activity against a highly virulent, drug-resistant strain of A. baumannii (AB5075). The most potent hit from this screen was an extract from the fungi Tolypocladium sp., which was found to produce pyridoxatin. Another active extract from the fungi Trichoderma deliquescens was characterized and yielded Trichokonins VII and Trichokonins VIII. Evaluation of pyridoxatin against A. baumannii (AB5075) in a broth microdilution assay revealed a minimum inhibitory concentration (MIC) of 38.0 µM, which was the strongest lead compared to the known antibiotic levofloxacin MIC of 27.7 µM. Mass spectrometry, Marfey’s analysis and nuclear magnetic resonance spectroscopy were utilized to confirm the structures of Trichokonins VII and Trichokonins VIII in comparison to previous reports. In an in vivo Galleria mellonella model, pyridoxatin tested at 150 mg/kg exhibited minimal toxicity (90% survival) and promising antimicrobial efficacy (50% survival) after 5 days. Trichokonins VII and Trichokonins VIII tested at 150 mg/kg were toxic, with 20% survival and 40% survival after 5 days, respectively. The findings of this project suggest that pyridoxatin may serve as a lead compound for the development of antimicrobials against A. baumannii. They also demonstrate the value of the phenotypic screening approach employed here to uncover diverse lead antimicrobial compounds.
A second approach to counteracting antimicrobial resistance was taken with a botanical extract mechanism of action classification prediction method that was developed using metabolomics in the model antimicrobial system of Hypericum calycinum (creeping St. John’s wort) against Methicillin-resistant Staphylococcus aureus (MRSA). Antimicrobial botanicals, such as H. calycinum, exhibit unique mechanisms of action due to a complex array of compounds and combination effects which challenges bacteria in developing resistance. MRSA strain USA300 LAC AH1263, a model organism we observed with susceptibility to H. calycinum, is a clinically relevant drug-resistant pathogen rapidly developing resistance. To evaluate these qualities, extracellular matrices were evaluated after incubation of MRSA with sublethal concentrations of six clinically relevant antibiotics representing three mechanisms of action, an H. calycinum extract, an H. calycinum active fraction, or the pure botanical compound hyperforin commonly found in Hypericum sp. Metabolomics, the study and comparison of small molecule distributions in biological samples, was selected to evaluate antimicrobial botanical mechanisms of action. Previously developed data processing techniques were refined for evaluating profiles of extracellular matrices and narrowing down features detected across all samples from 8,900 features to a list of 32 unique chemical footprints associated with mechanism of action. Further investigation of the identities of features detected in the chemical footprint and biochemical studies to confirm the antimicrobial mechanism of action of H. calycinum against MRSA are necessary to validate the model in predicting antimicrobial mechanism of action in botanical extracts.