Dissertation
DIVERSITY, ANTIMICROBIAL ACTIVITY AND METABOLOMICS STUDY OF MARINE MICROBES FROM PUERTO RICO AND HAWAII
Doctor of Philosophy (PhD), Washington State University
01/2018
Handle:
https://hdl.handle.net/2376/111137
Abstract
The need for novel antibacterial compounds has stimulated the exploration of marine environment for bioactive secondary metabolites. However, marine bacterial screening often rediscoveres known species, leading to an inefficient discovery process that is laborious and time consuming. The approach used in this investigation involves genus and possibly species level identification of a large number of samples from diverse locations, mass spectrometry-based finger printing/metabolomic analysis, study of the volatile metabolites and creation and expansion of database containing new actinobacterial species. In this study, replicate samples from 19 geographical locations of Puerto Rico and seven collection sites from Hawaii were analyzed to investigate the diversity of marine actinobacteria regarding antimicrobial properties and growth requirements for bacteria from similar tropical conditions. 16S rRNA sequencing on extracted, PCR amplified DNA samples helped identification at least to the genus level. Chemical fingerprinting, based on chemical analysis of secondary metabolites excreted during cultivation, is one possible discovery tool to explore the chemical picture of the produced set of metabolites without the individual isolation or any prior knowledge about the produced matabolites. Ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) was found to be an excellent tool for natural product analysis in this study. UPLC-MS analyzed ethyl acetate (EA) extracts of the bacterial samples with data being analyzed using Progenesis QI and Metaboanalyst software. Volatile metabolites from these bacterial strains were analyzed using GC-MS and ChromaTOF software. We used a nonderivatization approach, which allows faster analysis and successful observation of different classes of volatile organic compounds present in these samples. We tested antibacterial activity for each actinobacterial culture using both cell free supernatants and crude extracts against five different antibiotic resistant pathogens. Among 80 initial samples, nine showed antimicrobial activity against Acinetobacter baumannii (AB), Methicillin resistant Staphylococcus aureus (MRSA), and Enterococcus faecalis (EPH). One strain from Hawaii exhibited broad spectrum activity. Actinobacterial library creation was accomplished using Bruker Biotyper software from samples analyzed in a Bruker UltrafleXtreme, a state of the art MALDI-TOF/TOF system. Extension of the database using samples from different geographical locations, combined with antimicrobial activity testing results will make future identification of new, novel actinobacterial species routine.
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Details
- Title
- DIVERSITY, ANTIMICROBIAL ACTIVITY AND METABOLOMICS STUDY OF MARINE MICROBES FROM PUERTO RICO AND HAWAII
- Creators
- Ruhnaz Kashfi
- Contributors
- David Gang (Advisor)Douglas Call (Committee Member)Clifford Berkman (Committee Member)Ming Xian (Committee Member)
- Awarding Institution
- Washington State University
- Academic Unit
- Department of Chemistry
- Theses and Dissertations
- Doctor of Philosophy (PhD), Washington State University
- Number of pages
- 135
- Identifiers
- 99900581623701842
- Language
- English
- Resource Type
- Dissertation