Faculty of Science THE EXTRACTION AND ANALYSIS OF ANTIMICROBIAL SECONDARY METABOLITES PRODUCED BY CAVE STREPTOMYCES S1, S4, AND PM58B 2017 | RORY DAVID MCKERCHAR B.Sc. Honours thesis – Chemical Biology THE EXTRACTION AND ANALYSIS OF ANTIMICROBIAL SECONDARY METABOLITES PRODUCED BY CAVE STREPTOMYCES S1, S4, AND PM58B by RORY DAVID MCKERCHAR A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF BACHELOR OF SCIENCE (HONS.) in the DEPARTMENT OF BIOLOGICAL AND PHYSICAL SCIENCES CHEMICAL BIOLOGY Naowarat Cheeptham (Ph.D.) Thesis Co-supervisor, Dept. Biological Sciences Kingsley Donkor (Ph.D.) Thesis Co-supervisor, Dept. Physical Sciences Soumya Ghosh (Ph.D.) Thesis Co-supervisor, Dept. Biological Sciences Cynthia Ross Friedman (Ph.D) Examining Committee member, Dept. Biological Sciences Dated this 28th day of April 2017 in Kamloops, British Columbia, Canada © Rory McKerchar, 2017 ABSTRACT Antibiotic resistance is a growing problem as microorganisms exhibiting resistance to antibiotics of last resort have been reported in hospitals around the world. To respond to this problem, it is necessary to develop and discover novel antibacterial compounds. A rich reservoir of antibacterial compounds are microbial secondary metabolites, a structurally diverse group of molecules produced by microbes in response to changing environmental conditions providing some advantage to their producers. Particularly prolific producers of these compounds are bacteria of the group Streptomyces, these being responsible for a large portion of the naturally sourced antibacterial compounds used today. In order to access novel and untapped chemical diversity, the investigation of unexplored biological niches has become more prevalent, with the study of cave dwelling microorganisms producing promising leads. This study examines the antimicrobial secondary metabolites produced by three cave Streptomyces strains S1, S4, and PM58b for their previously described activity against target multi-drug resistant Escherichia coli and methicillin resistant Staphylococcus aureus with the goal of determining their molecular characteristics. To achieve this, three strains of Streptomyces were grown for periods of 10 to 30 days to induce the production of secondary metabolites. Bioactivity was confirmed in S1 and S4 by an agar-plug assay and was observed as a pigmented ring of inhibition after an extended period of incubation. Streptomyces strain S1 fermentation broth was extracted and analyzed by matrix assisted laser desorption ionization mass spectrometry for the preliminary identification of the antimicrobial compound present. Antimicrobial activity could not be isolated in a cell free environment from S1 fermentation broths; instead, the antimicrobials were produced on the assay plate. Differences were observed between the fermentation broth and sterile media mass spectra; however, a molecular mass could not be assigned to the putative antimicrobial compound. Thesis Co-supervisor: Associate Professor Naowarat Cheeptham (Ph.D.) Thesis Co-supervisor: Professor Kingsley Donkor (Ph.D.) Thesis Co-supervisor: Dr. Soumya Ghosh (Ph.D.) ii ACKNOWLEDGEMENTS I would like to thank Dr. Naowarat Cheeptham, Dr. Kingsley Donkor, and Dr. Soumya Ghosh for supervising my project and Dr. Cynthia Ross Friedman agreeing to act as my external honours committee member. I would like to acknowledge Dr. Cheeptham for providing guidance and insight in the culturing, maintenance and antimicrobial assaying of cave Streptomyces S1, S4, and PM58b and for always making time to talk when I had questions or ran in to problems. I would like to acknowledge Dr. Donkor for providing invaluable training on MALDI and other chemical instrumentation and for always having an open door. Dr. Soumya Ghosh’s help and direction in the laboratory, particularly in the designing and implementation of my bioassays for antimicrobial activity was crucial to my project. In addition to my supervisors and honours committee members, I would like to thank Dylan Ziegler for his critical eye, Dr. Don Nelson for his help in my literature search, and Dr. Louis Gosselin for his work as Honours Coordinator. Finally I would like to thank my friends and family for their love and support. iii TABLE OF CONTENTS ABSTRACT.................................................................................................................................................. ii ACKNOWLEDGEMENTS ......................................................................................................................... iii TABLE OF CONTENTS ............................................................................................................................. iv TABLE OF FIGURES .................................................................................................................................. v TABLE OF TABLES .................................................................................................................................. vi INTRODUCTION ........................................................................................................................................ 1 Antibiotic resistance.................................................................................................................................. 1 The Streptomyces lifecycle and the production of secondary metabolites................................................ 1 Accessing novel chemical diversity .......................................................................................................... 3 MALDI as a tool for the preliminary structural elucidation of secondary metabolites ............................ 4 Experiment goals and approach ................................................................................................................ 5 MATERIALS AND METHODS .................................................................................................................. 5 Growth of Cave Streptomyces Cultures.................................................................................................... 6 Bioassays for Antimicrobial Activity ....................................................................................................... 6 MALDI Sample Preparation ..................................................................................................................... 8 MALDI Sample Analysis ......................................................................................................................... 8 RESULTS ..................................................................................................................................................... 9 Growth of Cave Streptomyces Cultures.................................................................................................... 9 Bioassays for Antibacterial Activity ......................................................................................................... 9 MALDI Analysis of Fermentation Broth, and R2A Background ........................................................... 12 DISCUSSION ............................................................................................................................................. 13 CONCLUSIONS AND FUTURE WORK ................................................................................................. 15 LITERATURE CITED ............................................................................................................................... 16 APPENDICES .............................................................................................................................. 19 iv TABLE OF FIGURES Figure 1. Overview of experimental approach to the production and analysis of antimicrobial activity of cave Streptomyces strains S1, S4, and PM58b. ............................................................. 5 Figure 2. Antimicrobial activity plug assay of S1 and S4 fermentation broths grown for 8 and 30 day durations at 8°C against laboratory strains of E. coli and S. aureus. Plates were incubated at 37°C for a 7 day period ................................................................................................................. 11 Figure 3. Antimicrobial activity plug assay of S1 and S4 fermentation broths grown for 8 and 30 day durations at 8°C against laboratory strains of E. coli and S. aureus. Plates were incubated at 15°C for a 7 day period ................................................................................................................. 11 Figure 4. Antimicrobial activity plug assay of S1, S4, and PM58b fermentation broths grown for 10 and 30 day durations at 8°C and 15°C against a laboratory strain and MDR E. coli. Plates were incubated at 15°C for a 7 day period. ........................................................................................... 12 Figure 5. Antimicrobial activity plug assay of S1, S4, and PM58b fermentation broths grown for 10 and 30 day durations at 8°C and 15°C against a laboratory strain of S. aureus and MRSA. Plates were incubated at 15°C for a 7 day period. ................................................................................... 12 Figure 6. R2A background MS replicate 1 ................................................................................... 24 Figure 7. R2A background MS replicate 2 ................................................................................... 25 Figure 8. R2A background MS replicate 3 ................................................................................... 26 Figure 9. R2A background MS replicate 4 ................................................................................... 27 Figure 10. R2A background MS replicate 5 ................................................................................. 28 Figure 11. R2A background MS replicate 6 ................................................................................. 29 Figure 12. S1 30 day fermentation broth MS replicate 1 .............................................................. 30 Figure 13. S1 30 day fermentation broth MS replicate 2 .............................................................. 31 Figure 14. S1 30 day fermentation broth MS replicate 3 .............................................................. 32 Figure 15. S1 30 day fermentation broth MS replicate 4 .............................................................. 33 Figure 16. S1 30 day fermentation broth MS replicate 5 .............................................................. 34 Figure 17. S1 30 day fermentation broth MS replicate 6 .............................................................. 35 v TABLE OF TABLES Table 1. Compiled observations of antimicrobial activity of S1, S4 and PM58b at 15°C incubation temperatures under varying treatments. (+) designates bioactivity observed, (-) designates no bioactivity observed, and NA indicates that the combination of strains and treatments indicated were not tested. ............................................................................................................................. 10 Table 2. MALDI M/Z signal unique to the S1 30 day fermentation broth, obtained from the systematic comparison with R2A background spectra ................................................................. 21 Table 4. Tabulated R2A background peaks. Red shaded cells indicate peaks duplicated in more than one replicate. ......................................................................................................................... 22 Table 5. Tabulated 30 day fermentation broth peaks. Red shaded cells indicate peaks duplicated in more than one replicate. ................................................................................................................ 23 vi INTRODUCTION Antibiotic resistance The progressive spread of antibiotic resistance among pathogenic bacteria is a dire problem to which no clear solution has arisen, to the growing concern of public health authorities, governments, and medical professionals alike. The spread of these antibiotic resistant phenotypes is a clear example of artificial selection. This selective pressure is established as a consequence of the extensive use of antibiotics in agriculture, medicine, and daily life leading to the evolution of resistant strains of bacteria. Shortly after the advent of the widespread therapeutic use of antibiotics with the discovery and application of penicillin, antibiotic resistance has followed close behind with resistant strains becoming more and more prevalent (Davies and Davies 2010). The mechanisms underlying this resistance vary between the intrinsic resistances some groups of pathogenic microbes display to certain classes of antibiotics, to acquired resistances developed over time in response to the selective pressure placed on pathogenic bacteria due to the application of antibiotics (Blair et al 2015). Examples of these acquired resistances are drug efflux systems, antibiotic modifying enzymes, and changes to the cellular target itself (Blair et al. 2015). Adding to the problem of the emergence of antibiotic resistance is its capacity to spread between bacteria through the horizontal transfer of chromosomal and extrachromosomal genetic material leading to the emergence of multi-drug resistant (MDR) strains of bacteria (Cohen et al. 1972; Blahna et al. 2006; Davies and Davies 2010). In order to combat this spread of antibiotic resistance a multifaceted approach must be implemented through new restrictions on the availability and approved uses of existing antibiotics, the development of new analogues of existing antibiotics, and through the exploration of novel chemical space to identify new antibiotic compounds with both unique molecular scaffolds and mechanisms of action (Davies and Davies 2010). The Streptomyces lifecycle and the production of secondary metabolites Streptomyces have historically been a prolific microbial source of antimicrobial secondary metabolites, producing many of the antibiotics on the market today. These typically soil dwelling bacteria are Gram positive and undergo a complex life cycle, moving between vegetative and aerial hyphal states. Streptomyces grow from single spores into branching vegetative hyphae that elongate by apical tip extension forming a substrate mycelium (Sigle et al. 2015, Flardh and Buttner 2009). Responding to environmental cues such as nutrient depletion, the Streptomyces undergo a morphological differentiation producing aerial hyphae during which the secondary metabolism associated with the production of antimicrobial compounds is activated (Flardh and Buttner 2009; Sigle et al. 2015). The production of antimicrobial secondary metabolites in Streptomyces is regulated by a variety of factors, and the synthesis of these compounds can be induced in response to multiple stimuli. The presence of stimulatory precursor molecules can have an inductive effect on the synthesis of these secondary metabolites, as observed in the impact of lysine on the synthesis of cephamycin C in S. clavuligerus, or the role of valine in the synthesis of tylosin in S. fradiae (Nguyen et al. 1995; Demain 1998; Leite et al. 2013,). The synthesis of these antimicrobial compounds can also be due to the activity of endogenous auto-regulator molecules. These autoregulatory compounds take several forms one of which are the γ-butytrolactones. These compounds act as ligands to cytoplasmic regulatory proteins such as ArpA in S. griseus, binding and deactivating them, preventing their binding of the Streptomyces chromosomal DNA and fulfilling their role as repressors (Takano 2006). In S. griseus this ArpA deactivation allows for the synthesis of AdpA, a regulatory protein responsible for both the upregulation of the synthesis of secondary metabolism as well as the morphological differentiation of the bacteria (Bibb 2005). The morphological changes observed with activation of AdpA are the formation of aerial hyphae and their subsequent sporulation, a process promoted by lowered ATP levels. (Wolanski et al. 2012). The production of these autoregulatory compounds such as γ-butytrolactones are controlled by biosynthetic genes typically located nearby biosynthetic clusters responsible for secondary metabolites synthesis (Bentley et al. 2002; Liu et al. 2013). Other regulators in the production of secondary metabolites include the alarmone guanosine pentaphosphate (p)ppGpp, which in response to stresses such as heat shock or nutrient deprivation, modulates cellular metabolism and transcription in a concentration dependant manner initiating the stringent response (Hauryliuk et al. 2015). The cytosolic (p)ppGpp concentration is regulated by the ribosome associated enzyme Re1, which directly monitors the process of translation within the cell. When exposed to stress inducing conditions such as nutrient deprivation (p)ppGpp is synthesized, which through direct or indirect mechanisms, downregulates the production of ribosomal proteins while promoting the biosynthesis of amino acids and genes 2 associated with stationary phase processes such as secondary metabolism (Hesketh et al. 2007; Hauryliuk et al. 2015). Accessing novel chemical diversity Although the chemical diversity of Streptomyces secondary metabolism has been the subject of intense study, leading to the discovery of a multitude of anticancer, antifungal, and antibiotic compounds, science still has much to gain from further investigation of this genus. However, in order to achieve this goal scientists must employ new approaches and technologies. Through the development and application of metagenomic approaches to drug discovery, the identification of potential antimicrobial secondary metabolites through the detection and analysis of biosynthetic clusters associated with secondary metabolism progress has led to the discovery of new antimicrobial secondary metabolites (Handelsman 2004, Seipke 2015, Chu et al. 2016). In addition to these wide-net approaches to the discovery of new microbial sources of antibiotics, the exploration of new and understudied microbial niches such as the ocean or caves allows researchers to access new species of Streptomyces and potentially novel antibiotic secondary metabolites (Fielder et al. 2005, Cheeptham et al. 2013, Rateb et al. 2011). These understudied niches often represent extreme environments, with the cave environment being a clear example of this; having low levels of readily available organic carbon, little to no light, and high mineral and salt content (Ghosh et al. 2016). In these extreme environments, endogenous bacteria have evolved metabolic processes to access the limited resources available to them, which along with the rich diversity in the cave environment, contributes to the development of unique often species specific secondary metabolites (Bhullar 2012, Cheeptham et al. 2013, Cuezva et al. 2012). This study aims to explore the potentially novel chemical diversity associated with cave Streptomyces through the molecular characterization of previously described antimicrobial activity ascribed to Streptomyces strains isolated from Iron Curtain Cave near Chilliwack B.C. (S1 and S4) and from Helmcken Falls Cave in Wells Gray Provincial park near Clearwater B.C (PM58b). Helmcken Falls Cave has been extensively studied being identified as volcanic in origin, with subsequent modifications introduced through the activity of the nearby Murtle and Clearwater Rivers. The cave macroenvironment of Helmcken Falls Cave includes basalt walls and ceilings as well as deposits of fine sands and sediments (Cheeptham et al. 2013). Iron Curtain Cave located near Chilliwack B.C. although not nearly as well studied, is also a cave of volcanic origin (Mason et al. 2015). 3 MALDI as a tool for the preliminary structural elucidation of secondary metabolites In order to proceed with the drug discovery process, from the identification of an organism exhibiting antimicrobial activity, to the application of the bioactive compound in a clinical environment, the complete structural elucidation of the compound is necessary. A crucial step in this process is the determination of the molecular mass of the compound of interest by mass spectrometry, as this step facilitates further investigation by techniques such as IR and NMR, allowing for complete structural elucidation (Kind and Fiehn 2010). Mass spectrometry in conjunction with elemental analysis techniques such as energy dispersive X-ray spectroscopy, allows for the identification the molecular formula of the compound of interest and thus permits future studies of the connectivity and functionality of the compound by NMR and IR experiments (Kind and Fiehn 2010). In addition to this, dual mass spectroscopy (MS-MS) itself can be used to derive structural information of the compound of interest through its sequential and the prediction of fragmentation patterns (Cabrera 2006). Matrix assisted laser desorption ionization (MALDI) is a form of mass spectrometry that is typically used to analyze large molecules such as whole proteins or peptides. Although conventionally applied to these larger molecules, MALDI remains a valuable instrument in the analysis of smaller molecules such as those secondary metabolites being produced by Streptomyces (Cohen and Gusev 2002). MALDI-MS boasts high sensitivities and high mass resolutions while also being a high throughput technique allowing for the rapid analysis of many samples. In addition to this, MALDI is a soft ionization technique resulting in little to no fragmentation of target compounds. The consequence of this being, instead of producing charged fragments MALDI-MS tends to ionize the whole compound producing a quasi molecular ion (MH+) peak, providing information on the molecular weight of the compound allowing for greater ease the determination of the compound’s molecular formula. Although it benefits from being a high throughput technique capable of achieving high resolutions and sensitivities, MALDI suffers from the detection of matrix peaks at low m/z values such as those examined in this experiment (Cohen and Gusev 2002). In addition to this MALDI cannot be coupled directly to a chromatography column, preventing any separation of the compounds in the sample of interest prior to ionization. These problems can be addressed through the introduction of an ionization 4 suppressant such as cetyltrimethylammonium bromide (CTAB) and conducting a preparatory separation by column chromatography (Guo et al. 2002). Experiment goals and approach Using liquid-liquid extraction and MALDI-MS, this study aims to identify the antimicrobial secondary metabolites of cave Streptomyces strains S1, S4, and PM58b by assigning a molecular mass to these compounds. This is achieved by the nutrient deprivation of mature Streptomyces cultures through the growth of these bacteria in batch culture until fermentative conditions are achieved. The presence of antimicrobial activity is then to be confirmed through plug diffusion assays against multi-drug resistant and laboratory strains of S. aureus and E. coli. Once antimicrobial activity is established in the Streptomyces fermentation broths, liquid-liquid extraction of the responsible metabolite will be attempted with a variety of organic solvents, after which antimicrobial activity will be confirmed by further bioassays. Low m/z mass spectra will be obtained for these extracts and the original fermentation broths through MALDI-MS and antimicrobial activity will be correlated to the emergence and persistence of novel signals in the MS spectra. MATERIALS AND METHODS Growth Conditions for Streptomyces strains S1, S4, and PM58b established in R2A, V8, and Hickey Tresnar growth media Fermentative conditions established through the nutrient limitation of Streptomyces bacterial cultures Perform solvent extractions on Streptomyces fermentation broth and analyze these extracts using MALDI mass spectrometry Correlate the presence of antimicrobial compounds in fermentation broth extracts to specific peaks on mass spectra. Plug diffusion assays carried out to establish antimicrobial activity of Streptomyces fermentation broths against E. coli, MDR E. coli, S. aureus, and MRSA Figure 1. Overview of experimental approach to the production and analysis of antimicrobial activity of cave Streptomyces strains S1, S4, and PM58b. 5 Growth of Cave Streptomyces Cultures Streptomyces strains S1 and S4 were streaked for isolated colonies from stock plates onto Hickey-Tresnar (HT) Agar using aseptic technique and were then grown at 8°C and 15°C. These HT plates served as stock plates for work done using S1 and S4 in this experiment and were stored under refrigeration at 4°C. Isolated colonies of S1 and S4 were then aseptically transferred from these stock plates into 16x100 mm glass test tubes containing sterile 3 mL of R2A (S1) and V8 (S4) broth. Ten 3 mL cultures of each strain were prepared in this manner. From the twenty S1 and S4 cultures five of each strain were then grown at temperatures of 8°C and 15°C in shaking incubators for a period of 10 days. Following this period, the strains were assessed for antimicrobial activity, and analyzed by MALDI. These cultures were evaluated before conducting bioassays by Gram staining to ensure no contamination had occurred. Following these growth and antimicrobial activity assays S1 and S4 were grown at 8°C for durations up to 30 days under the above described conditions. These strains were then assessed for antimicrobial activity. Following this a 300 mL volume of sterile R2A broth was inoculated with S1 using 1.00 mL of a 3 mL five-day culture. This culture was incubated at 8°C for a period of 30 days after which extractions, bioassays for antimicrobial activity and mass spectrometry analyses were performed. Streptomyces strain PM58b was streaked for isolated colonies from a stock culture suspended in 30% glycerol stored at -20°C onto a HT agar plate where it was then grown at 8°C and 15°C. This HT plate served as a stock plate for the work done using PM58b for this study. The plate was stored under refrigeration at 4°C Isolated colonies were then aseptically transferred from the stock plate into16x100 mm glass test tubes containing sterile 3mL HT broth. These PM58b cultures were similarly fermented for durations of 10 to 30 days at temperatures of 8°C and 15°C after which the broth was bioassayed for antimicrobial activity. Bioassays for Antimicrobial Activity The Streptomyces strains were evaluated for antimicrobial activity using a plug diffusion assay described by S. Ghosh in a personal communication (2016). This assay was prepared using sterilized 225x225 mm polystyrene bioassay plates. These plates were sterilized using 1.5% Peroxyguard solution, 70% ethanol, and UV light exposure for a duration of 20 minutes. Following 6 this, 250 mL of autoclaved molten nutrient agar maintained at a temperature of 55°C was inoculated to give a concentration of 1x106 cells per mL of the target organism, and was delivered to the plate and allowed to solidify. The target organisms used for these assays were laboratory and drug resistant strains of S. aureus (MRSA-43300) and E. coli (15-104, 15-124, 15-318) which were provided by LifeLabs (Kamloops B.C.). Isolated colonies of these organisms were aseptically transferred from blood agar stock plates to 3 mL volumes of nutrient broth. These cultures were then incubated with shaking 100 rpm for a duration of 20-24 hours. The concentration of cells in the 3mL culture was then quantified by measuring the culture’s optical density (OD). This was done using a Pharmacia NovaSpecIII Spectrophotometer set at a wavelength of 600 nm and Plastibond 1.5 mL cuvettes to which 1.00 mL of culture was added. The spectrophotometer was blanked using sterile nutrient broth, after which OD measurements of the culture being assayed were made in triplicate. An average OD was taken, and using the assumption that an OD of 1.0 corresponds to a cellular concentration of 8x108 cells per mL, a volume of the 3 mL stock culture was delivered using a p1000 VWR micropipette and sterile disposable pipette tips to the molten agar to give a final concentration of 1x106 cells per mL in the nutrient agar. The nutrient agar was then delivered to the plate and allowed to solidify. Once the agar had solidified plugs were bored out of the agar using a flame sterilized bore with a 6 mm ID, producing wells with a volume of 100 µL. To these wells 100 µL of the material being assayed was delivered using a p200 VWR micropipette. As positive controls 100 µL volumes of Peroxyguard and 70% ethanol were used. For drug resistant organisms 6mm diameter diffusion disks impregnated with 30 µg of tetracycline produced by Beeton Dickinson and Company were used also used. Bioassay plates were incubated at 37°C, 25°C, and 15°C for a duration of 7 days, activity was evaluated daily. Alongside the various growth conditions assayed, assays were performed with fermentation broth supernatant samples, filtrate samples, heat shocked samples, and extracts. Supernatant samples were prepared by transferring 1.00 mL volumes of Streptomyces culture to autoclaved 1.5 mL Eppendorf tubes which were subsequently centrifuged at 14,000 rpm for a duration of 10 minutes on a Hermle Z 233 MK-2 Centrifuge. From these Eppendorf tubes 0.500 mL of the supernatant was then transferred to fresh, sterile 1.5 mL Eppendorf tubes from which it was delivered to the assay plate. Heat shocked samples were prepared by delivering 1.00 mL volumes of the Streptomyces broth to autoclaved 1.5 mL Eppendorf tubes which were then placed in a boiling water bath for a duration of 10 minutes. Filtered samples were obtained for S1 samples 7 by first centrifuging 1.00 mL volumes of fermentation broth as described above. The supernatants of these 1.00 mL volumes were then delivered to a sterile 5.0 cc Luer lock syringe using a p1000 VWR micropipette and was subsequently filtered through a 0.45 µm VWR cellulose acetate luer lock filter into sterile Eppendorf tubes from which the filtrate was delivered to the assay plate. Finally extract samples were prepared by centrifuging 30 mL volumes of the Streptomyces culture in 50 mL VWR pre-sterilized plastic centrifuge tubes at 5000 rpm in an IEC MP4 Centra centrifuge. The supernatant from these tubes was then poured through a 70µm nylon mesh Fischer Scientific cell strainer in to an autoclaved 250mL volumetric flask. Of this cell free filtrate 50 mL was measured out and transferred using a graduated cylinder into a 250 mL separatory funnel. Several extractions were performed, with the first using three 10 mL volumes of LC-MS grade hexane followed by three 10 mL volumes of LC-MS grade ethyl acetate. The fractions were combined and the solvents were evaporated in evaporating dishes under the fume hood. Upon the evaporation of the solvents the residues were dissolved in 2.00 mL of autoclaved 18 MΩ water, of which 1.5 mL was transferred into autoclaved Eppendorf tubes. Extractions were also attempted on the 70 µm filtrate using three 10 mL volumes of LC-MS grade ethyl acetate which was then similarly evaporated in the fumehood and subsequently dissolved in 2.00 mL of autoclaved 18 MΩ water. MALDI Sample Preparation Samples were prepared on a ground steel target plate for analysis by MALDI. The samples were prepared as per the dried droplet protocol described by Bruker Daltonics MALDI preparation protocols. Equal volumes of the fermentation broth sample and TA30 (30:70 (v/v) acetonitrile :0.1% trifluoracetic acid in 18 MΩ water) saturated with α-cyano-4-hydroxy cinnamic acid (HCCA) were premixed in a clean sterile Eppendorf tube. From this Eppendorf tube 1 µL of the sample-matrix solution was delivered directly to the ground steel plate. To ensure the spectra obtained were consistent and representative of the sample solution, fermentation broth samples were run in replicates of 6. The samples analyzed in this manner included, Sterile R2A, and 30 day S1 fermentation broth filtrate MALDI Sample Analysis Samples were analyzed using a Bruker MicroFlex LRF MALDI. The method used for this analysis was developed and implemented using Bruker Daltonics flexControl software (version 8 3.3.108.0). Compounds of m/z’s of 0 Da to 4000 Da were selected and evaluated with the response limited to a signal-to-noise threshold of 6.0 and over, a maximal peak number of 300, a maximal peak width of 0.2 m/z and a resolution higher than 100. Noise was reduced using a Tophat baseline subtraction algorithm. The sample and matrix were ionized using a 19.99 kV ion source with the laser set on the random walk setting. Samples were analyzed by summing up 800 satisfactory shots taken in 80 shot steps using a 60 Hz laser starting at 20% power before reaching maximal power at 80% power. The resultant spectra were analyzed using Flex Analysis software. Mass-to-charge values were copied into Microsoft Excel and the m/z values of the 30 day S1 fermentation broth were screened for peaks observed consistently in 2 or more of the 6 replicates analyzed within a difference of 0.005 m/z. These consistently observed peaks were then screened against all peaks observed in any of the R2A background spectra to identify signals present in the 30 day S1 fermentation broth but not present in the R2A background spectra. RESULTS Growth of Cave Streptomyces Cultures Strains S1, S4 and PM58b were observed to grow effectively at temperatures of 8°C and 15°C, growing faster at the 15°C condition. On HT agar S1 grew into large white filamentous colonies, S4 grew into flat gray filamentous colonies, and PM58b grew into large raised white filamentous colonies. In R2A broth S1 formed rounded filamentous pellet-like colonies, forming strand like structures on the walls of the test tube. S4 growing in V8 broth was observed to form smaller flocculant, forming filamentous strand like structures on the walls of the test tube. PM58b formed white filamentous colonies in HT broth forming strand like structures on the walls of the test tube. In all three cultures, growth was evaluated visually as the pellet and flocculant nature of the Streptomyces strains prevented optical density measurements and made dilution and plating approaches non-representative of the actual number of cells in the culture. As the fermentation broths aged, S1 and S4 cultures developed a brown pigmentation which became more pronounced as the culture grew older; this pigmentation was not observed in PM58b. Bioassays for Antibacterial Activity Antimicrobial activity was observed in S1 and inconsistently in S4 against S. aureus, E. coli, MRSA and MDR E. coli. No antimicrobial activity was observed in the PM58b fermentation 9 broth. The antimicrobial activity was observed after a 10 day fermentation period up until 30 days of fermentation in S1, beyond which assays were not performed. In S4 activity was only observed after an extended period of nutrient limitation, with the effects being observed after a 30 day fermentation. Antimicrobial activity presented in both strains as a zone of inhibition surrounding the plug to which the fermentation broth was delivered. In addition to being devoid of the cloudiness characteristic of growth of the target organisms, these zones of inhibition were pigmented a dark brown. In the plug holes from which these regions of antimicrobial activity radiated from, Streptomyces growth was observed. This antimicrobial activity was observed when the bioassay plates were incubated at 15°C however this activity was not present when higher temperatures (37°C) were used. The antimicrobial activity developed over the duration of the incubation of the bioassay plate with activity first appearing as a small pigmented zone of inhibition after day 2, and developing into a larger clearer pigmented zone of inhibition after day 7. Antimicrobial activity was observed in the supernatants of S1 and S4; however, this activity was accompanied with growth of the Streptomyces on the walls of the plug holes. Antimicrobial activity was not observed in heat shocked samples in either strain. Antimicrobial activity was also not observed in the 0.35 µm filtered S1 fermentation broth. Neither the hexane nor the ethyl acetate extracts exhibited antimicrobial activity against the target organisms. Similarly, when the aqueous fraction of the extracted broth was tested, no activity was observed. When assays were performed with the unextracted fermentation broth supernatant, which had previously only been filtered through a 70 µm cell strainer antimicrobial activity was observed along with growth of S1 within the agar plug hole. Table 1. Compiled observations of antimicrobial activity of S1, S4 and PM58b at 15°C incubation temperatures under varying treatments. (+) designates bioactivity observed, (-) designates no bioactivity observed, and NA indicates that the combination of strains and treatments indicated were not tested. Strain S1 S4 PM58 b 8 day 10 day 30 day Super natant 0.35µm Filtrate Ethyl Acetate Extract Hexane Extract Aqueous Fraction 70µm strained broth Heat Shock + - + - + + + + NA NA NA NA + NA - - - - - NA NA NA NA NA - 10 Figure 2. Antimicrobial activity plug assay of S1 and S4 fermentation broths grown for 8 and 30 day durations at 8°C against laboratory strains of E. coli and S. aureus. Plates were incubated at 37°C for a 7 day period Figure 3. Antimicrobial activity plug assay of S1 and S4 fermentation broths grown for 8 and 30 day durations at 8°C against laboratory strains of E. coli and S. aureus. Plates were incubated at 15°C for a 7 day period 11 Figure 4. Antimicrobial activity plug assay of S1, S4, and PM58b fermentation broths grown for 10 and 30 day durations at 8°C and 15°C against a laboratory strain and MDR E. coli. Plates were incubated at 15°C for a 7 day period. Figure 5. Antimicrobial activity plug assay of S1, S4, and PM58b fermentation broths grown for 10 and 30 day durations at 8°C and 15°C against a laboratory strain of S. aureus and MRSA. Plates were incubated at 15°C for a 7 day period. MALDI Analysis of Fermentation Broth, and R2A Background From the comparison of the R2A background and the 30 day S1 fermentation broth 25 consistently observed signals were identified as being present in the fermentation broth but absent in the R2A background spectra. These unique signals ranged from 236.881 to 623.675 m/z. These signals were tabulated in the Appendix. 12 DISCUSSION The results of the accumulated bioassays conducted using S1 indicate that the fermentative conditions previously described failed to elicit antimicrobial activity in the cell free fermentation broth. No antimicrobial activity was observed when Streptomyces cells were not present or failed to thrive in the plugs of the bioassay plate. This lack of activity in the absence of cells was indicated by the results obtained from filtering the supernatant of the 30 day S1 fermentation broth through a 0.35 µm cellulose acetate filter. This filtration removed the Streptomyces cells, leaving an inactive cell free aqueous fermentation broth. This result pointed towards the bioactive compound responsible for the antimicrobial activity observed in bioassays of the cell containing culture being absent or present in concentrations too low to have an observable effect in the cell free broth. This was partially supported by the results of bioassays conducted using heat shocked supernatant. The heat shocking of the Streptomyces fermentation broth at 100°C for 10 minutes likely killed the majority of cells persisting in the supernatant leading to no Streptomyces growth on the bioassay plate and therefore no antimicrobial activity. However, the treatment that killed the Streptomyces present in the supernatant may have also degraded the antimicrobial compound, leading to a loss of bioactivity and making this result inconclusive on its own. More direct support for the conclusion that the antimicrobial compound was not substantially present in solution are the results of assays conducted using the re-dissolved residues of organic extracts and the aqueous component of the organic extract. The extraction of a 50 mL volume of strained S1 fermentation broth supernatant using both hexane and ethyl acetate likely killed any persister cells in the broth, leading to no live Streptomyces in the re-dissolved organic residue or in the aqueous fraction. While this treatment likely killed the persisting Streptomyces cells it would have had minimal impact on any potentially bioactive compounds in solution, and should have concentrated any that partitioned into the ethyl acetate or hexane fractions. Assaying these re-dissolved extracts, the aqueous fraction they were extracted from and an unextracted portion of the strained fermentation broth supernatant showed no antimicrobial activity but for the unextracted broth after an extended incubation. This indicates that the antimicrobial compound of interest was not produced in solution and was instead being produced on the bioassay plate. The antimicrobial activity exhibited by Streptomyces strain S4 presented in a similar manner as S1, showing activity against E. coli and S. aureus, in conjunction with the spread of dark brown pigment that developed and spread over 13 time. The antimicrobial activity attributed to S4, however, was obtained inconsistently, occurring in only two separate sets of bioassays in which 30-day old small test tube scale fermentation broths were used. This inconsistency and loss of activity when the process was scaled up to larger volumes prevented further exploration of the antimicrobial activity. PM58b exhibited no activity at all in this experiment, which similarly prevented further exploration. Observations on the effects of the incubation conditions and duration on the antimicrobial activity of S1 indicate that the antimicrobial compound was produced on the plate. Firstly, the development of antimicrobial activity over the duration of the incubation of the bioassay plates indicates that the compound was produced in increasing concentrations on the bioassay plate as the Streptomyces present grew and developed. The degree of antimicrobial activity was observed to progress over time increasing in intensity and in the radius of the zone of inhibition. This observation contrasted sharply with the behaviour of the positive controls; Peroxyguard and tetracycline, which involved finite amounts of each compound, and presented as clearly delineated zones of inhibition that developed shortly after the delivery of the control to the bioassay plate. The lack of antimicrobial activity at 37°C despite the presence of Streptomyces cells in the bioassay wells supports the idea that the antimicrobial compound is produced on the plate as a result of Streptomyces cells growth. This is because the cave Streptomyces S1were unable to grow at the 37°C incubation temperature, and as such the antimicrobial was not produced. Despite not being secreted into the fermentation broth, the progressive diffusion displayed by the antimicrobial compounds produced by S1 suggests that the antimicrobial compound was secreted and not surface bound to the microbe as Streptomyces are non-motile organisms. In addition to this, the extent of the diffusion of the antimicrobial compound on the bioassay plates suggests that the compound is water soluble due to its capacity to diffuse through the aqueous agar. The lack of activity in the cell free broth may indicate that the nutrient depleted conditions in the S1 fermentation broth failed to induce the production of antimicrobial secondary metabolites, the antimicrobial activity observed on the bioassay plates after an extended incubation may have provided these nutrient limited conditions stimulating the production of these metabolites. This achievement of nutrient depleted conditions stimulating the production of secondary metabolism on the bioassay plate but not in the fermentation broth is consistent with the observations of gradual diffusion and development of the antimicrobial compound on the 14 bioassay plate. Similarly, the observation that no antibacterial activity was exhibited in cell free variations of the fermentation broth also supports this conclusion, as in the absence of cells, the induction of secondary metabolism cannot be achieved. An alternative interpretation of the results above describes an antimicrobial compound being produced by Streptomyces S1 in a manner that is attached to the outer cell surface, preventing the isolation of antimicrobial activity in a cell free environment. This interpretation of the experimental data would explain the diffusion of the antimicrobial activity as the result of the growth of hyphae through the agar giving this diffuse zone of inhibition. This interpretation could be tested through the bioassaying of a fermentation broth of S1 after the constituent bacteria’s outer cellular surface has been disrupted. The results of the MALDI-MS analysis of the 30-day fermentation broth produced signals not observed in the R2A background media. This is to be expected as the result of normal microbial metabolism however due to the inability to correlate any antimicrobial activity to the cell free fermentation broth. The spectra obtained appeared quite noisy potentially due to signals detected from the ionized HCCA matrix and matrix adducts of sodium or potassium. CONCLUSIONS AND FUTURE WORK The results of this research leave many questions to be answered, and could open up doors to new potential research questions. The observation that antimicrobial activity consistently failed to be observed in the cell free fermentation broth despite being produced on the plate opens the opportunity to further fine-tune fermentation conditions so as to produce the antimicrobial secondary metabolites in solution allowing for their extraction and for further analysis to take place. In future applications of MALDI to the analysis of such complex mixtures blanks should include several replicates of matrix only prepared with hard water to evaluate the presence of any matrix salts to prevent these signals from confounding results. 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Open Biology. 2, 120097 (2012). 18 APPENDICES Oxoid Nutrient Agar Media 1.0g/L Lab-Lemco powder 2.0g/L Yeast extract 5.0g/L Peptone 5.0g/L Sodium chloride 15g/L Agar pH 7.4 Oxoid Nutrient Broth Media 1.0g/L Lab-Lemco powder 2.0g/L Yeast extract 5.0g/L Peptone 5.0g/L Sodium chloride pH 7.4 R2A Broth Media 0.500g/L Casein acid hydrolysate 0.500g/L Yeast extract 0.500g/L Peptone 0.500g/L Dextrose 0.500g/L Starch, soluble 0.300g/L Dipotassium phosphate 0.024g/L Magnesium sulphate 19 0.300g/L Sodium pyruvate pH 7.2 V8 Broth Media 200mL reduced salt V8 juice supernatant (centrifuged at 5000rpm for 10 minutes) 3.0g/L Calcium carbonate pH 7.4 Hickey Tresnar Broth Media 1.0g/L Yeast extract 1.0g/L Beef extract 2.0g/L N2 Amine 10.0g/L Dextrin pH 7.5 Table 2. Reagents used. Reagent and Grade Distributor HPLC grade hexane Sigma Aldrich LC grade ethyl acetate Sigma Aldrich Reagent grade trifluoroacetic acid Sigma Aldrich Reagent grade acetonitrile Sigma Aldrich 18MΩ water - Reagent grade 2-Propanol Fischer Scientific Α-Cyano-4-hydroxycinnamic acid Bruker Daltonics 20 Table 2. MALDI M/Z signal unique to the S1 30 day fermentation broth, obtained from the systematic comparison with R2A background spectra Unique 30 Day Broth MS Signals 236.881 244.738 246.72 247.572 250.708 253.578 256.465 262.4 263.463 264.344 274.14 276.246 285.965 293.681 301.795 301.816 311.354 327.263 329.239 338.211 372.196 373.339 420.557 608.043 623.675 21 Table 3. Tabulated R2A background peaks. Red shaded cells indicate peaks duplicated in more than one replicate. R2A Background R2A Background R2A Background R2A Background R2A Background R2A Background R2A background 1 2 3 4 5 6 Consistent 203.492 203.492 203.492 203.492 203.492 203.492 203.492 219.299 219.352 219.305 219.318 219.437 219.305 219.305 227.819 228.085 227.819 227.819 219.437 228.085 219.437 230.817 230.823 230.822 230.822 230.823 230.823 227.819 231.642 231.918 231.918 231.908 231.918 231.918 228.085 232.746 234.953 232.746 232.746 232.746 232.746 230.822 233.642 236.87 233.756 234.952 234.952 234.953 231.918 234.952 237.727 234.952 235.784 235.784 235.784 232.746 236.616 238.835 236.616 236.696 236.708 236.72 234.952 237.727 239.68 237.728 237.728 237.727 237.727 235.784 238.563 242.755 238.563 238.563 238.68 238.673 236.616 242.483 243.609 239.679 239.679 239.68 239.68 237.727 258.205 258.338 242.483 242.483 242.605 242.593 238.563 259.186 259.368 243.608 243.608 243.608 243.609 239.68 260.243 260.243 254.443 258.205 253.576 258.205 242.483 262.289 262.29 258.205 259.368 254.443 259.368 243.608 273.844 274.138 259.363 260.243 258.205 260.243 254.443 274.842 275.043 260.243 262.29 259.368 262.29 258.205 275.945 275.945 262.29 273.948 260.243 273.977 259.368 276.915 277.148 273.844 275.043 262.29 275.043 260.243 301.504 301.504 275.038 275.945 271.155 275.945 262.29 314.376 314.56 275.945 276.989 273.977 277.148 273.844 315.333 315.532 276.947 278.054 275.043 278.054 273.977 341.567 339.208 281.996 281.996 275.945 301.504 275.043 354.475 341.901 301.504 301.504 276.976 314.56 275.945 384.978 382.289 314.553 314.553 278.054 315.532 277.148 385.746 384.979 315.528 315.523 301.504 341.901 278.054 419.432 386.054 341.891 341.891 314.558 354.475 281.996 422.429 387.128 382.118 354.475 315.528 379.263 301.504 445.716 403.845 384.979 382.119 341.894 382.118 314.56 465.219 406.789 386.053 384.978 354.475 384.979 315.532 466.48 419.432 403.841 386.053 369.035 386.054 341.891 481.272 422.436 419.432 403.844 382.119 392.903 341.901 602.126 446.1 422.435 419.432 384.978 394.715 354.475 657.949 449.984 445.851 445.717 386.052 403.845 382.118 673.766 465.687 465.672 449.978 403.844 406.789 384.979 689.785 466.607 466.48 465.684 406.77 408.636 386.053 481.273 481.273 466.589 419.432 419.432 403.844 497.132 483.29 481.273 422.435 422.435 406.789 658.424 674.22 658.424 446.083 446.083 419.432 674.248 689.785 674.245 449.98 465.687 422.435 690.267 705.812 689.785 465.684 466.48 446.083 706.005 693.188 466.585 481.273 465.684 706.002 481.273 658.425 465.687 658.418 674.248 466.48 674.246 689.785 481.273 689.785 706.005 658.424 692.221 674.248 706.002 689.785 708.937 706.002 22 Table 4. Tabulated 30 day fermentation broth peaks. Yellow shaded cells indicate peaks duplicated in more than one replicate. Broth 1 Broth 2 236.887 239.825 242.762 244.738 246.72 250.708 256.465 258.385 259.368 262.443 268.474 274.14 275.044 276.247 277.15 278.055 281.996 285.966 301.816 311.657 314.567 315.534 372.528 373.354 399.084 419.432 420.557 449.984 465.687 225.645 230.823 236.888 242.76 246.72 258.374 259.368 262.582 268.182 274.141 275.043 276.246 277.15 281.996 292.129 301.819 311.674 314.567 315.534 317.473 338.211 339.216 372.547 481.273 689.784 Broth 3 230.823 236.883 237.727 239.945 242.756 244.738 246.72 247.572 250.708 253.578 256.465 258.333 259.369 262.4 263.462 264.344 274.14 275.044 276.231 278.055 285.965 301.801 311.526 314.567 315.534 327.263 329.239 372.525 373.339 608.041 624.131 Broth 4 236.881 237.727 239.953 244.737 246.72 247.572 250.708 253.578 256.465 258.325 259.369 262.397 263.463 264.344 270.261 274.138 275.044 276.246 277.15 278.055 285.965 301.795 311.512 314.567 315.534 327.263 329.239 353.789 372.519 373.339 403.845 409.746 419.432 481.273 608.043 624.119 706.005 Broth 5 236.616 237.728 246.714 247.572 258.312 262.29 263.169 264.344 274.142 275.044 276.246 277.15 278.055 293.681 301.504 311.354 314.556 315.523 338.21 338.993 372.196 373.252 403.844 404.58 419.432 420.545 421.307 481.273 497.131 598.951 623.675 631.569 674.249 690.271 706.005 721.928 Consistent Signals 236.738 230.823 237.728 236.881 242.483 236.888 246.719 237.727 247.572 242.76 253.578 244.738 256.465 246.72 258.205 247.572 262.29 250.708 264.344 253.578 274.13 256.465 275.043 259.368 276.246 262.29 277.15 262.4 278.055 263.463 293.681 264.344 301.504 274.14 311.354 275.043 314.566 276.246 315.534 277.15 338.198 278.055 372.196 281.996 373.31 285.965 403.845 293.681 409.744 301.504 419.77 301.795 420.557 301.816 481.273 311.354 497.131 314.567 623.675 315.534 327.263 329.239 338.211 372.196 373.339 403.845 419.432 420.557 481.273 497.131 608.043 623.675 706.005 Broth 6 23 Figure 6. R2A background MS replicate 1 24 Figure 7. R2A background MS replicate 2 25 Figure 8 R2A background MS replicate 3 26 Figure 9 R2A background MS replicate 4 27 . Figure 10 R2A background MS replicate 5 28 Figure 11 R2A background MS replicate 6 29 Figure 12 S1 30 day fermentation broth MS replicate 1 30 Figure 13 S1 30 day fermentation broth MS replicate 2 31 Figure 14 S1 30 day fermentation broth MS replicate 3 32 Figure 15 S1 30 day fermentation broth MS replicate 4 33 Figure 16 S1 30 day fermentation broth MS replicate 5 34 Figure 17 S1 30 day fermentation broth MS replicate 6 35