Faculty of Science SCREENING OF CAVE BACTERIA FOR ANTIMICROBIAL ACTIVITY AGAINST PSEUDOMONAS AERUGINOSA BIOFILMS 2015 | COHORD MASON B.Sc. Honours thesis Screening of Cave Bacteria for Antimicrobial Activity against Pseudomonas aeruginosa Biofilms by COHORD MASON A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF BACHELOR OF SCIENCE (HONS.) in the DEPARTMENT OF BIOLOGICAL SCIENCES (Cellular, Molecular, and Microbial Biology) This thesis has been accepted as conforming to the required standards by: Naowarat Cheeptham (Ph.D.), Thesis Supervisor, Dept. Biological Sciences (TRU) Ken Wagner (M.D., F.R.C.P.), Co-Supervisor, Dept. Biological Sciences (TRU) Mario Jacques (Ph.D.), Co-Supervisor, Dept. Pathology and Microbiology (U of Montreal) Cynthia Ross Friedman (Ph.D.), Co-supervisor, Dept. Biological Sciences (TRU) Joanna Urban (RT, MSc.), Thesis Committee member, Dept. Biological Sciences (TRU) Dated this 14th day of May, 2015, in Kamloops, British Columbia, Canada i ABSTRACT The bacterium Pseudomonas aeruginosa may grow in a biofilm structure, which can be up to 1000 times more resistant to antibiotics compared to planktonic isolates. The P. aeruginosa biofilms have serious implications with regard to infection, especially in individuals with weakened immune defenses such as burn and cystic fibrosis patients. Antibiotic studies are usually based on planktonic antibiotic susceptibility results, so the treatment may be less effective when used in patients. The aim of this study was to further screen previously studied cave bacterial isolates with potential activity and determine their antimicrobial capabilities against P. aeruginosa biofilms. Three strains; A1A3, RA003 and 58B were cultured in different media over 10 days, with collections of supernatant on days 2, 4, 6, 8, and 10. The MBEC P&G Assay device was used to culture P. aeruginosa biofilms, which were then exposed to the collected supernatants. After exposure, the surviving biofilms were recovered, and spot plated in order to measure any inhibition of P. aeruginosa. Dilution and spot plating were also used to enumerate surviving cells, and give a percent survival quantification of antimicrobial activity. As well, Kirby-Bauer disc diffusion assays were used to determine the cave isolates’ inhibitory effect on planktonic cells. Scanning electron microscopy was used to examine biofilms structures and 16S rRNA sequencing was used to identify cave bacterial isolates. The biofilms showed a noticeably decreased percent survival when exposed to the cave isolate supernatant. The cave isolate 58B showed to be very promising, demonstrating significant reduction in the surviving biofilm cells. This study shows that cave bacteria produce antimicrobials that are effective against pathogenic bacteria even in a biofilm structure. ii ACKNOWLEDGEMENTS I would like to thank TRU UREAP for funding the pilot study for this project. I would also like to thank my supervisors Drs. Naowarat Cheeptham, Ken Wagner, Mario Jacques, and Cynthia Ross Friedman for guidance with this research, Joanna Urban for acting as a committee member for my Honour’s thesis defense, May Al-Fouadi for her lab assistance, and Baylee Out for assistance with the sequence alignment. I would like to especially thank my family and friends for providing support throughout this research project. iii Contents ABSTRACT.............................................................................................................................. ii ACKNOWLEDGEMENTS ..................................................................................................... iii LIST OF FIGURES ................................................................................................................. vi LIST OF TABLES ................................................................................................................... vi INTRODUCTION .................................................................................................................... 1 Antibiotic Resistance ............................................................................................................ 1 Biofilms................................................................................................................................. 2 Pseudomonas aeruginosa Pathogen ..................................................................................... 3 Cave Bacteria ........................................................................................................................ 4 Activation of Antimicrobial Compounds with Ultraviolet Light ......................................... 4 Objective ............................................................................................................................... 5 MATERIALS AND METHODS .............................................................................................. 6 Culturing of Pseudomonas aeruginosa Biofilms in MBEC Device ..................................... 6 Scanning Electron Microscopy Preparation and Set-Up: Viewing Pseudomonas aeruginosa............................................................................................................................. 8 Culturing of Cave Bacteria Isolates ...................................................................................... 8 Antimicrobial Exposure ...................................................................................................... 10 Neutralization of the Antimicrobial and Recovery of Surviving Bacteria ......................... 12 Kirby-Bauer Disc Diffusion Assay to Determine Planktonic Antimicrobial Activity ....... 14 iv Molecular Identification of Pseudomonas aeruginosa and Cave Bacterial Isolates by 16S rRNA gene sequencing ....................................................................................................... 16 RESULTS AND DISCUSSION ............................................................................................. 17 Scanning Electron Microscopy for Viewing Pseudomonas aeruginosa ............................ 17 Antimicrobial Activity of Cave Bacterial Isolates Observed with Spot Plating................. 19 Antimicrobial Activity of Cave Bacterial Isolates Determined with Calculation of CFU/mL ............................................................................................................................................. 22 The Antibiotic and Disinfectant Susceptibilities of P. aeruginosa in a Biofilm ................ 26 Antimicrobial Activity of cave Bacterial Isolates on Planktonic P. aeruginosa ................ 28 The Antibiotic and Disinfectant Susceptibilities of P. aeruginosa in a Planktonic Form .. 29 Antimicrobial Activity as Compared to Previous Studies with Same Isolates ................... 30 Identities of Cave Bacterial Isolates and P. aeruginosa ..................................................... 32 CONCLUSIONS AND FUTURE WORK ............................................................................. 35 LITERATURE CITED ........................................................................................................... 38 APPENDIX A-MEDIA .......................................................................................................... 42 APPENDIX B-RAW DATA .................................................................................................. 44 APPENDIX C-CONTIGUOUS SEQUENCES OF BACTERIA........................................... 54 v LIST OF FIGURES Figure 1: Flow diagram representing the sample collection, and exposure of P. aeruginosa to the cave bacterial supernatant samples. .................................................................................... 6 Figure 2: The MBEC P&G device used to culture P. aeruginosa into a biofilm formation. The device is composed of a 96 well plate with a matching 96 peg lid, with each peg fitting into a well. ................................................................................................................................. 7 Figure 3: Diagram of rinsing procedure used for preparation for the initial removal, challenge plate and recovery plate (Ceri et al., 1999). ............................................................................ 10 Figure 4: Challenge plate schematic containing the 3 cave bacteria isolate samples with UV and Non-UV treatments cultured in R2A, ISP2, and V8 media. There are 2 antibiotics used in four concentrations: Tetracycline HCl at 120, 60, 30, and 10 μg/mL and Ciprofloxacin at 10, 5, 2.5, and 0.5 μg/mL. Also there is 2% Virkon controls, 0.1 N HCl (or sterile water) controls and sterile media controls (R2A, ISP2, and V8). .................................................................... 11 Figure 5: Example of Large TSA plates used to visually determine growth of P. aeruginosa (Picture after 18 hours of incubation in 35oC). ....................................................................... 13 Figure 6: Example of large square agar plates with discs containing cave bacteria isolate samples, and controls used to assess the antimicrobial activity of each sample. The diameters of the dark inhibitory zones that encircle each disc were measured to assess the antimicrobial activity..................................................................................................................................... 16 Figure 7: SEM images of P. aeruginosa Biofilms viewed with Zeiss Evo LS SEM; A) Image of Biofilm showing planktonic cells to mature biofilms; B) Image of planktonic cells C) Image of mature biofilm with mushroom like colonies( left arrow indicates water channels and right arrow indicates mushroom like colonies) ................................................................ 18 Figure 8: The number of samples produced by the cave bacteria that showed antimicrobial activity against P. aeruginosa in a biofilm or planktonic form. ............................................. 20 Figure 9: Results of spot plating surviving P. aeruginosa biofilms onto TSA plates after exposure to Day 8 samples: A) A1A3 V8 Non-UV B) RA003 R2A Non-UV C) RA003 ISP2 Non-UV D) RA003 V8 Non-UV E) RA003 V8 UV F) 58B R2A Non-UV G) 58B V8 NonUV H) 58B V8 UV I) Sterile TSB. The numbers 1, 2, and 3 denote replicate number of each sample. .................................................................................................................................... 22 Figure 10: The average survival of P. aeruginosa cells (in CFU/mL) compared to the TSB control after exposure to Day 8 samples (n=3). The error bars indicate standard error of the mean. ....................................................................................................................................... 24 LIST OF TABLES Table 1: Table of each sample from Day 8 that had an antimicrobial effect and P. aeruginosa biofilms. The surviving cells (CFU/mL) from the biofilms after exposure were calculated from the spot plating the cells. The three replicates were used to find the average CFU/mL and then the average percent survival by taking the sample average and dividing it by the TSB control. The two sample T test (n=3) was used to find if the surviving cell percentage was the same between biofilms exposed to the cave isolate samples and the TSB control. .. 25 vi Table 2: The surviving cells (CFU/mL) of the P. aeruginosa biofilms after exposure to Ciprofloxacin HCl (day 10 exposures). .................................................................................. 28 Table 3: Identification of the organisms used according to 16S rRNA gene sequencing ...... 33 vii INTRODUCTION Antibiotic Resistance The decline in the discovery of novel antibiotics and rising of antibiotic resistance in pathogens has made the discovery of new ones important. Antibiotics are essential to the medical field, and help us combat bacterial and fungal infections, which, according to the World Health Organization, cause 16.2% of the world’s deaths per year. The antibiotics work by interacting with a bacterium or fungi, and disrupting a certain pathway resulting in the organism’s destruction. When the antibiotics are first introduced, there may be a small proportion of resistant bacteria in patients, but these bacteria are not yet problematic. Then the number of resistant bactereria may rise to a level where the antibiotic has reduced efficiency in the human population, resulting in antibiotic resistant bacteria (Coates and Hu, 2007). Antibiotic resistance precedes clinical use, and can be found in nature (Hall and Barlow, 2004), but has increased due to the over-use in bacterial infection therapies (Fernández et al., 2011). From the overuse of antibiotics, some bacteria have seemingly undergone an accelerated evolution, and have increased success of surviving antibiotic treatment. This has allowed some bacterial species to become “multi-drug resistant” (MDR). Through continuous exposure, some bacteria have become resistant to these compounds and employ many different defenses. These defenses may be passed through horizontal gene transfer, conjugation, or mutations leading to decreased susceptibility (Baquero, 2001). Antibiotic resistance may be from bacteria having enzymes that break down the antibiotic, reducing the permeability of the bacterial cell, or from changing the target of the antibiotic. 1 An interesting example of a resistant defense is biofilms that are produced by some bacterial species. Biofilms Biofilms are communities of bacteria encased in a self-synthesized polymeric matrix that may adhere to biotic or abiotic surfaces (Hall-Stoodley et al., 2004). The cells of the biofilm are typically encased in a matrix of polysaccharides, extracellular DNA (eDNA), and proteins (Flemming and Wingender, 2010; Yang et al., 2012). Bacteria are usually thought of as planktonic, single cell organisms, but they predominantly live in multicellular biofilms in the environment. Environmental stress results in dysregulation of multiple regulatory systems, and the cells will attach to a surface in order to resist the environmental change. The formed mature biofilm may release some cells in a dispersive stage in order to colonize new areas (Costerton et al., 1999; Hall-Stoodley et al., 2004). Biofilms may have water-filled channels that allow for increased nutrient supply. The matrix may also contain extracellular enzymes, and cells may show enhanced toxic compound excretion, changed metabolic processes and differentiated phenotypes. That is, the biofilm creates a gradient of nutrients and oxygen with inner cells having a decreased metabolism and cell division relative to the peripheral cells (Costerton et al., 1999; Flemming and Wingender, 2010; Hall-Stoodley et al., 2004). This mode of growth protects the bacteria from eradication via desiccation, nutrient deprivation and antibiotic treatment (Gaddy and Actis, 2009). Bacteria associated with biofilms may be up to 1000 times more resistant to antibiotics compared to planktonic cells, and enables cells in biofilms to persist despite intensive antibiotic therapy (Gaddy and Actis, 2009; Mah and O’Toole, 2001). Antibiotic 2 therapy may even trigger biofilm formation if the antibiotics are at a concentration below the minimum inhibitory concentration (known as sub-MIC). Bacteria may be exposed to subMIC at the beginning and end of antibiotic therapy. Also, the colony of cells may be thick enough such that only low amounts of the antibiotic actually make it to inner cells, resulting in sub-MIC (Mah and O’Toole, 2001; Singh et al., 2010). The bacterium Pseudomonas aeruginosa is a clinically important organism because it forms resilient biofilms. Pseudomonas aeruginosa Pathogen Pseudomonas aeruginosa is a motile, Gram-negative, opportunistic pathogen that is prevalent in the medical hospital environment. The pathogen is common in respiratory infections and urinary tract infections, and is especially implicated in patients with weakened defenses like burn and cystic fibrosis (CF) patients (Morita et al., 2014). The bacterium P. aeruginosa is a metabolically versatile microbe that can survive in many different environments. It can grow in aerobic and anaerobic conditions and may produce a multitude of different virulence factors (Schurek et al., 2012). The pathogenic bacteria may possess flagella and type IV pili that can function in adhesion and motility as well as initiation of an inflammatory response (Gellatly and Hancock, 2013). Also, Type 3 secretion systems inject toxins directly into host cells and breach epithelial cell layers (Hauser, 2009). The bacterium P. aeruginosa may also produce proteases that are able to degrade immunoglobulins and fibrin, leading to tissue damage (Kipnis et al., 2006). Some other virulence factors include exotoxin A, lipases, pyocyanin, and iron chelators (Gellatly and Hancock, 2013). As well as possessing many virulence factors, P. aeruginosa is also resistant to many antibiotics. The bacterium has intrinsic resistance to many antimicrobials because of an outer-membrane barrier, multidrug efflux pumps and endogenous antimicrobial inactivation (Poole, 2011). 3 Most importantly the bacterium can produce a robust biofilm, which is thought to further its antibiotic resistance ability. This resistance by P. aeruginosa has allowed it to persist despite tough antibiotic therapies so there is a need for new antibiotics. Cave Bacteria Caves are extreme habits that may house new microorganisms, which can potentially produce many new bioactive compounds. The caves are usually nutrient deprived so these organisms must have adapted in order to survive in such an environment (Cheeptham et al., 2013). In particular, Gram-positive, filamentous actinomycetes have been proven to be a new source of antibiotics (Genilloud et al., 2011). Out of 22,500 biologically active compounds, 45% are from actinomycetes (Bérdy, 2005), and the species Streptomyces accounts for 70% of the total antibiotic production (Lam, 2006). Cave actinomycetes are of particular interest because of the unique environment that they live in. It is reasonable to hypothesize that microorganisms living in caves are subject to regressive evolution, where some non-essential traits are lost over time. The extreme environment would result in bacteria losing some genes but developing new ones with different metabolic pathways. These new pathways could also result in new secondary metabolites, which could be important in drug discovery (Cheeptham et al., 2013). Activation of Antimicrobial Compounds with Ultraviolet Light There have been many studies on the activation of natural compounds with either full spectrum sunlight or ultraviolet light. These studies include the activation of biocidal compounds such as plant extracts, cow urine and even some antibiotics (Cheeptham and Towers, 2002; Upadhyay et al., 2010; Yuan et al., 2011). In most cases, organic compounds 4 absorb UV light and results in photolytic degradation, but this can be gradual which may result in new conformations of the compound (Kim and Tanaka, 2009). In one such study UV was used on antibiotics like ciprofloxacin, which resulted in an increase of activity against to Vibrio fisheri (Yuan et al., 2011). Our study also used UV light to potentially activate natural compounds to determine if they have any increased antimicrobial properties against a biofilm. Objective The objective of this research was to test the hypothesis that secondary metabolites produced by cave actinomycetes will have antimicrobial and/or antibiofilm properties against Pseudomonas aeruginosa in a biofilm. This includes determining the most effective cave bacteria isolate against P. aeruginosa, as well as the most proficient growth conditions to produce that effective secondary metabolite. Three cave bacteria isolates (A1A3, RA003, and 58B) previously showing activity were cultured for 10 days in different fermentation media, with samples taken on each day throughout the fermentation period. Collected samples were used to treat P. aeruginosa biofilms using a 96 well plate assay. The antimicrobial activity was measured by using dilution series and viable cell counts. A Kirby-Bauer disc diffusion assay was used to determine the effect of the supernatant on planktonic cells. Scanning electron microscopy was used to examine the biofilms presence and structure. Antibiotic susceptibilities of biofilms and planktonic cells compared the difference in resistances to the antibiotics. Lastly 16s rRNA sequencing identified the cave isolates’ genera. 5 MATERIALS AND METHODS Figure 1: Flow diagram representing the sample collection, and exposure of P. aeruginosa to the cave bacterial supernatant samples. Culturing of Pseudomonas aeruginosa Biofilms in MBEC Device The MBEC P&G assay device was used to culture the P. aeruginosa in a biofilm formation according to Standard Test Method for Testing Disinfectant Efficiacy against Pseudomonas aeruginosa Biofilm using the MBEC Assay (2014). The pathogenic bacteria P. aeruginosa (lab strain with unknown strain number) was first subcultured onto a trypticase soy agar (TSA) (Appendix A) plate using the four way streak method for isolated colonies. The plate was incubated for 18 hours at 35oC, and then inspected after the incubation to ensure a pure culture. Once the TSA plate had pure colonies of P. aeruginosa, the biofilm culturing began. 3.0 mL of sterile trypticase soy broth (TSB) was put into a sterile 16x100 mm glass test tube. A sterile cotton swab was used to collect P. aeruginosa colonies from the TSA plate. The swab with the bacteria was then deposited into the glass tube with TSB. The sample was 6 brought to a 1.0 McFarland standard using the deposited bacteria. This was achieved by taking a 500 uL sample and using a spectrophotometer at 600 nm (1.0 Mcfarland standard; OD=0.257 at 600 nm). Once the sample was at a 1.0 McFarland standard, 1.0 mL was added to 29 mL of TSB in a 45 mL plastic sterile centrifuge tube (this resulted in a 30x dilution) and served as the inoculum. A sterile MBEC P&G assay 96 well plate (Innovotech, Suite 101, 2011-94 St. Edmonton, AB, Canada) (Figure 2) was then removed from the packaging. All 96 wells were aseptically filled with 150 μL of the inoculum previously prepared. The MBEC device was then put in a 35oC incubator with an orbital shaker at 100 rpm for 18 hours. After 18 hours, the MBEC device was removed from the incubator. Figure 2: The MBEC P&G device used to culture P. aeruginosa into a biofilm formation. The device is composed of a 96 well plate with a matching 96 peg lid, with each peg fitting into a well. 7 Scanning Electron Microscopy Preparation and Set-Up: Viewing Pseudomonas aeruginosa The pegs with the P. aeruginosa biofilms were then removed from the lid of the MBEC device. This was achieved by using sterilized (flamed) needle nose pliers, grasping the pegs closed to the lid, and breaking the peg from the lid. The pegs were then put in 0.9% saline solution for one minute to remove any planktonic cells. The pegs were then placed horizontally on a stub (Specimen mount, pin type, slotted head) with carbon tape, and were loaded into the chamber of the Zeiss LS EVO SEM. The operating conditions used were: the vacuum at EP 60 Pa, accelerating voltage at 20 kV, filament amperage 1.684 A, spot size 100 pA, working distance 12 mm and the VPSE G3 detector (personal communication with Dr. Cindy Ross Friedman). The resulting images were adjusted for brightness and contrast with Microsoft Word tools to achieve the best picture quality. Culturing of Cave Bacteria Isolates The screening of cave bacteria isolates used three different isolates that previously showed activity against P. aeruginosa in a biofilm formation (Mason, 2015). The three strains used were RA003, A1A3, and 58B because they showed the ability to inhibit biofilms. These samples originally came from a volcanic cave at Wells Gray Provincial Park in British Columbia. Some samples came from agar plates stored in a 4oC fridge, and some from storage in 10% glycerol in 96 well plates at a temperature of -70oC (cryopreservation). Samples on agar plates had colonies removed with a sterile loop, and deposited on fresh Hick-Tresner (HT) (Appendix A) agar plates. The deposited sample was streaked for isolated 8 colonies using the 4-way streak method in order to achieve pure colonies of the cave isolates. Samples form cryopreservation were allowed to thaw slightly to liquid, then 10 μL samples were removed, deposited on HT agar plated and streaked for confluent growth. These samples were then used to inoculate new HT agar plates, which were then streaked for isolated colonies in order to obtain pure cultures. These plates were incubated at 25oC for 7 to 10 days and checked for pure colonies. Plates with mixed growth were then cultured again to obtain pure samples. Plates with pure growth were parafilmed and stored at 4oC for later use. Colony morphology, Gram-staining and microscopy were used to confirm samples were isolates from the previous studies. The three isolates were then used to inoculate test tubes containing different broth media. The 16X100 mm test tubes were filled with 6 mL of either V-8 juice, International Streptomyces Project #2 (ISP2), or R2A fermentation media (Appendix A), then autoclaved at 121oC for 15 minutes. Then 0.5x0.5 cm square plugs were aseptically cut out of the agar plates containing colonies of the desired cave bacterial isolate. These plugs were then put in the appropriate test tubes and cultured at 25oC for 10 days with 100 rpm of agitation in an orbital shaker (New Brunswick Scientific Innova 42). The samples of the supernatant from each tube were taken at days 2, 4, 6, 8, and 10. The test tubes were removed from the incubator and put on a sterilized bench. Then 800 μL samples were removed from each tube with care taken not to gather any cellular debris present. The 800 μL samples were then put into 1.0 mL Eppendorf tubes and stored at -20oC until used. 9 Antimicrobial Exposure The MBEC device was used to perform antimicrobial exposures. The MBEC device was removed from the incubator; the peg lid was removed and rinsed once in 0.9% saline solution for 1 minute (Figure 3). The supernatant collected from the cave bacteria samples was thawed in the 1.0 mL Eppendorf tubes. The tubes were then put in a centrifuge at 5000 rpm for 2 minutes. Any remaining cell debris would then be forced to the bottom of the Eppendorf tube. A sterile Nunc 96 well plate (washed with 2% Virkon, 70% ethanol, then exposed for 20 minutes to UV light) was used for the antimicrobial exposure and deemed the “challenge plate.” The wells for the UV treated samples were filled with 200 μL of the supernatant from the appropriate cave bacteria sample with triplicates of each. The 96 well plate with the supernatant was then exposed for 30 minutes to UV light (254 nm) in a Labconco Class II biological safety cabinet. Figure 3: Diagram of rinsing procedure used for preparation for the initial removal, challenge plate and recovery plate (Ceri et al., 1999). The Non-UV samples and controls were then added to the 96 well plates. The wells for the Non-UV treated samples were filled with 200 μL of the supernatant from the appropriate cave bacteria sample. The controls were then added to the appropriate wells: Sterile TSB, 2% Virkon, sterile V8, sterile R2A, sterile ISP2 and sterile 1.0 N HCl or water 10 (Figure 4). The growth controls would be those biofilms exposed to only sterile TSB, and the fermentation media controls would be biofilms exposed to the sterile V8, R2A, ISP2, and 0.1 N HCl or water. The Positive control was the biofilms that were exposed to a 2% Virkon solution (A disinfectant effective against P. aeruginosa), as well as the antibiotics Tetracycline HCl (120, 60, 30, and 10 μg/mL) and Ciprofloxacin in 0.1 N HCl (10, 5, 2.5 and 0.5 μg/mL) or Ciprofloxacin HCl (10, 5, 2.5 and 0.5 μg/mL). Figure 4: Challenge plate schematic containing the 3 cave bacteria isolate samples with UV and Non-UV treatments cultured in R2A, ISP2, and V8 media. There are 2 antibiotics used in four concentrations: Tetracycline HCl at 120, 60, 30, and 10 μg/mL and Ciprofloxacin at 10, 5, 2.5, and 0.5 μg/mL. Also there is 2% Virkon controls, 0.1 N HCl (or sterile water) controls and sterile media controls (R2A, ISP2, and V8). The peg lid from the MBEC device (after rinse [Figure 3]) was placed in the challenge plate with the appropriate orientation. The peg lid with the Challenge plate was then incubated at 35oC (no shaking) for 18 hours after which the MBEC device was removed from the incubator. 11 Neutralization of the Antimicrobial and Recovery of Surviving Bacteria After the peg lid and challenge plate was removed from the incubator, the peg lid was rinsed twice in 0.9% saline solution for 1 minute each wash. A sterile Nunc 96 well plate had all 96 wells aseptically filled with 200 μL of D/E neutralizing broth (Appendix A). The peg lid was then placed in the D/E neutralizing broth for 1 hour to neutralize the antimicrobials. The peg lid was then placed in a sterile 96 well plate with all 96 wells filled with 200 μL of TSB (deemed “recovery plate”). The peg lid and recovery plate were then sonicated for 30 minutes to remove the biofilm from the pegs. Two large plates (Figure 5), each filled with 250 mL of TSA, were used to check the results of the antimicrobial exposure. Each plate was divided into 49 sections, each with the appropriate sample area (Non-UV treated, UV treated, controls etc.). Then, 10 μL from each well of the recovery plate was spot plated onto the appropriately labelled square. The two TSA plates were then incubated at 35oC for 18 hours, and the recovery plate was put in a 4oC fridge. 12 Figure 5: Example of Large TSA plates used to visually determine growth of P. aeruginosa (Picture after 18 hours of incubation in 35oC). The TSA plates after 18 hours were removed and visually inspected to determine if any samples had no growth or reduced growth. Any samples showing no growth or reduced growth were spot plated to enumerate the colonies and to determine the CFU/mL. The recovery plate with the same sample on the TSA plate was used to make 4 dilutions from 10-1 to 10-4. The dilutions then had 10 uL removed and spot plated onto TSA plates. As well the P. aeruginosa growth controls (only exposed to TSB) and sterile media controls (R2A, ISP2, and V8) were also diluted the same and spot plated onto TSA plates. These TSA plates were then incubated at 35oC for 18 hours. After 18 hours, the TSA plates were removed from the incubator. The TSA plates had the cell colonies enumerated in order to determine the CFU/mL. The CFU/mL for each sample was then subjected to a 2 sample T test with pooled variances (after testing data for normality 13 and equal variance) to determine if the difference was significant. A P value of <0.05 was considered significant. A sample that showed 0 CFU/mL of surviving bacteria was designated bactericidal or if the concentration was known, then it was the minimum biofilm eradication concentration (MBEC). Kirby-Bauer Disc Diffusion Assay to Determine Planktonic Antimicrobial Activity The standard Kirby-Bauer (KB) disc diffusion assay (Bauer et al., 1966; Wilkins et al., 1972) was used to determine the antimicrobial activity of the cave isolates against planktonic P. aeruginosa. The discs used were 8 mm Advantec paper discs (Tokyo, Japan) that had previously been autoclaved to sterilize them. Each cave bacterial isolate sample was put on the discs in replicates of three. The antibiotics tetracycline HCl (120, 60, 30, and 10 μg/mL) and ciprofloxacin (10, 5, 2.5 and 0.5 μg/mL) or ciprofloxacin HCl (10, 5, 2.5 and 0.5 μg/mL) as well as 2% Virkon were used as positive controls. Additionally, sterile growth media (R2A, ISP2 and V8), sterile TSB, and sterile 0.1 N HCl were also used as negative controls. Each of the UV treated cave bacteria isolate samples had 80 μL pipetted onto each disc, and the discs were then exposed for 30 minutes to UV light (254 nm) in a Labconco Class II biological safety cabinet. Then 80 μL of the non-UV samples and controls were then pipetted onto the discs. All the discs including UV, non-UV and control discs were allowed to dry for 45 minutes. Large square agar plates were used to carry out the Kirby-Bauer disc diffusion assay. Two plates were used to accommodate all cave bacterial isolates and controls. Two 250 mL flasks were prepared containing 200 mL each of molten Trypticase soy agar which was 14 autoclaved at 121oC for 15 minutes. A sterile cotton swab was used to collect P. aeruginosa colonies from the TSA plate. During this time two 16x100 mm test tubes had 5 mL of sterile TSB pipetted into them. Then P. aeruginosa colonies were collected from a previously cultured TSA plate and deposited into each of the test tubes. Each test tube was brought to a 1.0 McFarland standard using the deposited bacteria. This was achieved by taking a 500 uL sample and using a spectrophotometer at 600 nm (1.0 Mcfarland standard; OD=0.257 at 600 nm). After the molten Trypticase soy agar flasks were autoclaved, the flasks were put into a 55oC water bath so the molten agar to equilibrate to the same temperature. At this point 2 mL of the prepared P. aeruginosa inoculum from one test tube was deposited into the 200 μL of molten TS agar (1% v/v), and then repeated with the other test tube and flask. Each flask was then poured into sterile large square plates and allowed to solidify. The previously prepared discs were then deposited onto the inoculated large square agar plates in alternating rows of 8 and 7 discs (Figure 6). The plates were then incubated at 35oC for 18 hours, removed and inhibitory zones were measured in millimeters. A sample showing an inhibitory zone was designated as having an inhibitory effect and if the concentration was known, then it was the minimum inhibitory concentration (MIC). 15 Figure 6: Example of large square agar plates with discs containing cave bacteria isolate samples, and controls used to assess the antimicrobial activity of each sample. The diameters of the dark inhibitory zones that encircle each disc were measured to assess the antimicrobial activity. Molecular Identification of Pseudomonas aeruginosa and Cave Bacterial Isolates by 16S rRNA gene sequencing In order to prepare for the gene sequencing, fresh agar plates were used to culture the cave bacterial isolates as well as P. aeruginosa. The cave bacteria were inoculated onto new HT agar plates, which were then streaked for isolated colonies in order to obtain pure cultures. These plates were incubated at 25oC for 7 days and checked for pure colonies. P. aeruginosa (lab strain with unknown strain number) was cultured onto a trypticase soy agar plate using the four way streak method for isolated colonies. The plate was incubated for 18 hours at 35oC, and then inspected after the incubation to ensure that it was a pure culture. The plates 16 were then sent to Seoul, Korea for 16S rRNA gene sequencing by Macrogen. Sequencing used 518F/800R and 27F/1492R primers and performed with Big Dye terminator cycle sequencing products, which were resolved on Applied Biosystem model 3730XL automated DNA sequencing system at Macrogen. The resulting 16S rRNA sequences were compared with the GenBank database by using BLAST. RESULTS AND DISCUSSION Scanning Electron Microscopy for Viewing Pseudomonas aeruginosa The structure of the P. aeruginosa biofilm was able to be viewed with the Zeiss Evo LS SEM. This helped establish that the P. aeruginosa was indeed in a biofilm formation and that characteristics such as increased antibiotic resistance would be observed in susceptibility tests. The images produced revealed a gradient of single planktonic cells to what appears to be mature biofilms. The initial attached cells were near the top of the peg close to the surface of the culture media. The biofilms started at the bottom of the peg and continued to where the individual cells initially attached. As seen in Figure 7A, starting in the top left of the image, small dots are visible, which would characterize the single planktonic P. aeruginosa. Figure 7B shows planktonic cells that appear to have the intermediate shape known as coccobacillus which would represent P. aeruginosa. Continuing from the top left to bottom right of Figure 7A, there seems to be aggregation of cells into clusters characteristic of a biofilm, more specifically microcolonies. In this same image, channels are apparent throughout the microcolony and mature biofilm portion. These water filled channels enable enhanced access 17 of nutrients throughout the biofilm and allow thicker biofilm areas to have enough resources to survive (Costerton et al., 1999; Flemming and Wingender, 2010; Hall-Stoodley et al., 2004). Also, Figure 7C shows a much more rounded structure which appears to be slightly raised from the rest of the biofilm. This mushroom-like structure is usually indicative of a mature portion of the biofilm and has been shown to have high amounts of eDNA (AllesenHolm et al., 2006). Overall the SEM under partial vacuum produced images with good resolution which could be used to identify components of the biofilm. Similarly, Alhede et al., (2012) used environmental conditions with the SEM to obtain similar images to this study. They used biofilm samples placed in the SEM chamber with minimal preparation and used a partial vacuum. Figure 7: SEM images of P. aeruginosa Biofilms viewed with Zeiss Evo LS SEM; A) Image of Biofilm showing planktonic cells to mature biofilms; B) Image of planktonic cells C) Image of mature biofilm with mushroom like colonies( left arrow indicates water channels and right arrow indicates mushroom like colonies) 18 Antimicrobial Activity of Cave Bacterial Isolates Observed with Spot Plating All three strains, A1A3, RA003 and 58B showed inhibitory activity against Pseudomonas aeruginosa biofilms. Similar to results seen in a previous study (Mason, 2015), these isolates did not show any inhibitory activity until day 8. Previously these strains did not produce metabolites that inhibited biofilms until day 7, and lost the activity by day 11 (Mason, 2015). Furthermore the Kirby-Bauer disc diffusion assay showed similar results with none of the cave isolates exhibiting inhibition of P. aeruginosa with sample days 2, 4, 6, 8, and 10. The sample days 2, 4, 6, and 10 had no indication of reduced growth after plating out surviving biofilms so those days did not have the colony numbers enumerated or CFU/mL calculated. Day 8 visually showed reduced or changed colony morphology compared to the growth controls (R2A, ISP2, V8 and TSB). The recovery 96 well plated was used to make dilutions, spot plate the dilutions, and enumerate the colonies to determine the CFU/mL (Figure 8). 19 Number of samples with Effect on P. aeruginosa Antimicrobial Effect on Biofilm Inhibitory Effect on Planktonic culture 9 8 7 6 5 4 3 2 1 0 Day 2 Day 4 Day 6 Day 8 Day 10 Sample Day Figure 8: The number of samples produced by the cave bacteria that showed antimicrobial activity against P. aeruginosa in a biofilm or planktonic form. The day 8 samples showed all three strains (A1A3, RA003, and 58B) having an inhibitory effect against the biofilms. This agrees with the preliminary study where all three had inhibitory activity at day 7(Mason, 2015). The colony spots of the cave isolate samples were compared to the TSB control (biofilm only exposed to TSB) colony spots (Figure 9 I). A1A3 cultured in V8, and not exposed to UV (Non-UV) reduced the colony spot in replicate 1. However the replicates 2 and 3 did not show reduced or changed colony morphology compared to the controls (Figure 9 A). The cave isolate RA003 cultured in R2A Non-UV also showed some inhibition in replicate 1 with the colony spot having colonies spread out rather than one solid colony spot. However the replicates 2 and 3 did not show visible inhibition (Figure 9 B). The cave isolate RA003 cultured in ISP2 Non-UV also had a reduced 20 colony spot, with only a few smaller colonies present. The replicates 2 and 3 had no visible reduction in the colony spot and probably were not affected (Figure 9 C). The cave isolate RA003 cultured in V8 Non-UV showed a smaller colony spot overall indicating some inhibitory effect. The replicates 2 and 3 did not visibly show any inhibition (Figure 9 D). The cave isolate RA003 V8 with UV exposure showed some inhibition with the colony spot of replicate 3 showing a lighter colony colour possibly from reduced cells. The replicates 1 and 2 did not show any inhibition (Figure 9 E). The cave isolate sample 58B R2A Non-UV completely eradicated the biofilm in replicate 1 evident by the no colony spot. The replicates 2 and 3 did not show any visible colony spot reduction (Figure 9 F). The sample 58B V8 Non-UV had replicate 1 having a colony spot with small spread out colonies rather than one large colony spot indicating biofilm inhibition. The replicates 2 and 3 did not visibly show any reduction in the colony spot (Figure 9 G). Lastly the sample 58B V8 UV had replicates 2 and 3 showing no colony spot indicating complete biofilm eradication. However, replicate 1 did not show any inhibition (Figure 9 H) (Appendix B). 21 Figure 9: Results of spot plating surviving P. aeruginosa biofilms onto TSA plates after exposure to Day 8 samples: A) A1A3 V8 Non-UV B) RA003 R2A Non-UV C) RA003 ISP2 Non-UV D) RA003 V8 Non-UV E) RA003 V8 UV F) 58B R2A Non-UV G) 58B V8 Non-UV H) 58B V8 UV I) Sterile TSB. The numbers 1, 2, and 3 denote replicate number of each sample. Antimicrobial Activity of Cave Bacterial Isolates Determined with Calculation of CFU/mL The day 8 samples that visually showed reduction of the biofilm demonstrated by an abnormal or absent colony spot on the TSA plates had the CFU/mL calculated. The calculated CFU/mL for each set of sample replicates agreed with the visual colony spot on the TSA plates. That is, if the colony spot was reduced, than the CFU/mL for that replicate reflected a decreased CFU/mL when compared to the TSB control. The calculated CFU/mL for each growth control (R2A, ISP2 and V8) was compared to the TSB control using a 2 sample T-test to confirm that the media had no effect on the P. aeruginosa biofilms. The Pvalue for each comparison of media to TSB control was greater than 0.05 meaning that there was no significant difference. This means that the R2A, ISP2 and V8 media did not affect the 22 P. aeruginosa biofilms. The cave bacteria isolate samples were also compared in a similar fashion with the use of a 2 sample T-test. The samples exposed to A1A3 V8 Non-UV, RA003 R2A, ISP2 and V8 Non-UV, 58B R2A Non-UV and RA003 V8 UV all had P-values greater than 0.05 and that the difference in CFU/mL was not significant. The biofilms exposed to 58B V8 Non-UV and 58B V8 UV both showed a significant difference (P-value less than 0.05) between the CFU/mL for these samples compared to the TSB control (Table 1). This would suggest that there was some metabolite in the supernatant samples from the 58B cave isolate cultured in V8 medium and was active with and without UV exposure. The cave isolate 58B cultured in V8 also showed antimicrobial activity against P. aeruginosa biofilms (Figure 10). 23 Control Non-UV Treated Samples UV Treated Samples Average Survival of P. aeruginosa after Exposure to Day 8 Samples (CFU/mL) 25000000 20000000 15000000 10000000 5000000 0 Sterile Trypticase Soy Broth (TSB) A1A3 V8 Non- RA003 R2A Non- RA003 ISP2 Non- RA003 V8 Non- 58B R2A Non-UV 58B V8 Non-UV UV UV UV UV RA003 V8 UV 58B V8 UV Biofilm Exposed to: Figure 10: The average survival of P. aeruginosa cells (in CFU/mL) compared to the TSB control after exposure to Day 8 samples (n=3). The error bars indicate standard error of the mean. The samples exposed to A1A3 V8 Non-UV, RA003 R2A, ISP2 and V8 Non-UV, 58B R2A Non-UV and RA003 V8 UV did not show significant reduction of CFU/mL compared to the TSB control. However, at least one replicate of each sample did show a noticeably reduced CFU/mL. A1A3 V8 Non-UV replicate #1 had a CFU/mL of 2.8x104 which is noticeably different than the TSB average of 7.6x106 CFU/mL. RA003 in R2A, ISP2 and V8 Non-UV also had one replicate with an observable difference; 3.5x104, 2.8x104, and 4.5x104 CFU/mL respectively. As well 58B R2A Non-UV had one replicate of 4.5x105 CFU/mL which differs slightly from the TSB control of 7.6x106 CFU/mL (Table 1). This 24 could be due the fact that the three replicates for each sample were actually from separate tubes. The tubes were in the same conditions (temperature, humidity, oxygen, etc.) but small differences inside the tubes could have resulted in different secondary metabolite production, having different antimicrobial efficiency. Table 1: Table of each sample from Day 8 that had an antimicrobial effect and P. aeruginosa biofilms. The surviving cells (CFU/mL) from the biofilms after exposure were calculated from the spot plating the cells. The three replicates were used to find the average CFU/mL and then the average percent survival by taking the sample average and dividing it by the TSB control. The two sample T test (n=3) was used to find if the surviving cell percentage was the same between biofilms exposed to the cave isolate samples and the TSB control. Sample A1A3 V8 Non-UV RA003 R2A Non-UV RA003 ISP2 Non-UV RA003 V8 NonUV 58B R2A Non-UV 58B V8 Non-UV RA003 V8 UV 58B V8 UV R2A sterile ISP2 sterile Surviving Cells After Exposure (CFU/mL) Average CFU/mL Average % survival P value comparing sample to TSB control Significantly different than TSB control Replicate Replicate Replicate #1 #2 #3 2.8x104 2.7x107 1.2x107 1.3x107 171.2 0.560 No 3.5x104 6.9x106 2.2x107 9.6x106 127.0 0.783 No 2.8x104 3.1x105 6.5x106 2.3x106 30.0 0.132 No 4.5x104 3.4x106 5.4x106 2.9x106 38.8 0.102 No 4.5x105 7.5x106 6.1x106 4.7x106 61.6 0.315 No 7.8x105 1.2x106 1.8x106 1.3x106 16.6 0.001 Yes 6.8x106 5.8x106 7.4x106 6.7x106 87.7 0.228 No 1.1x106 0 0 3.5x105 4.6 0.001 Yes 6.1x106 6.2x106 6.8x106 6.4x106 83.8 0.075 No 7.8x106 6.2x106 5.9x106 6.6x106 87.3 0.269 No 25 V8 sterile TSB 6.4x106 7.8x106 7.0x106 7.1x106 93.0 0.420 No 7.1x106 7.3x106 8.4x106 7.6x106 N/A N/A N/A The Antibiotic and Disinfectant Susceptibilities of P. aeruginosa in a Biofilm The controls used for sample days 2, 4, 6, 8, and 10 included 2% Virkon and antibiotics which had different effects in this sample day. The positive control, 2% Virkon, had biocidal properties, and resulted in the complete eradication of the biofilm. After the spot plating the Virkon exposed samples, there was no visible growth on the TSA plate. This would suggest that Virkon completely killed the P. aeruginosa biofilm. The biocidal activity of Virkon has previously been studied (Hernndez et al., 2000), and found that it is effective after 5 minutes of contact time with bacteria. Hernndez et al., (2000) found that 5 minutes of contact to P. aeruginosa had complete biocidal activity, and therefore 0 CFU/mL. Furthermore this study showed that P. aeruginosa biofilms exposed to Virkon for 18 hours also resulted in 0 CFU/mL. Tetracycline HCl (120, 60, 30, and 10 μg/mL) and ciprofloxacin (10, 5, 2.5 and 0.5 μg/mL) were both used but showed different effectiveness. The antibiotic tetracycline HCl at a 120, 60, 30, and 10 μg/mL had no inhibitory effect against the P. aeruginosa biofilms. The colony spot of the biofilm exposed to tetracycline even at a concentration as high as 120 μg/mL did not show any sign of inhibition. Tetracycline is a broad spectrum antibiotic with activity against Gram positive and Gram negative bacteria. It works by inhibiting protein synthesis by preventing the attachment aminoacyl-tRNA to the ribosomal acceptor (A) site (Chopra and Roberts, 2001). However P. aeruginosa is intrinsically resistant to tetracycline due the MexAB/MexXY efflux systems. 26 The efflux pumps remove the molecule from the cell before it can reach toxic concentrations (Morita et al., 2001) therefore tetracycline is generally not used against P. aeruginosa. The antibiotic ciprofloxacin (dissolved in 0.1 N HCl) at 10, 5, 2.5 and 0.5 μg/mL was used as a positive control on days 2, 4, 6, and 8. This antibiotic showed no colony spots on the TSA plates indicating biocidal activity. However, the control with only 0.1 N HCl also had no colony spot indicating complete biocidal activity. This would suggest that it was not the ciprofloxacin alone that was able to eradicate the P. aeruginosa biofilms. Most likely the 0.1 N HCl did not having enough nutrients to sustain the cells. The Kirby-Bauer disc diffusion assay (discussed later) showed that 0.1 N HCl had no inhibitory effect on P. aeruginosa in a planktonic state. However with the day 10 samples, ciprofloxacin HCl was used (dissolved in water) as a positive control at 10, 5, 2.5 and 0.5 μg/mL. When ciprofloxacin HCl was used against P. aeruginosa biofilms, it had biocidal effects. Since ciprofloxacin is dissolved in water, it was most likely the antibiotic that had biofilm eradication effect. At concentrations of 10 and 5 μg/mL it had complete biocidal activity and eradicated the biofilm resulting in 0 CFU/mL. The antibiotic is a fluoroquinolone and targets DNA gyrase which then inhibits cell division (Poole, 2011). The 5 μg/mL concentration represents the MBEC of ciprofloxacin HCl on P. aeruginosa. This MBEC agrees with other values found which put the MBEC of P. aeruginosa (ATCC 27853) at 4 μg/mL (Ceri et al., 1999) but a more recent study put it in the range of 0.25-8 μg/mL (same strain) (Dosler and Karaaslan, 2014). 27 Table 2: The surviving cells (CFU/mL) of the P. aeruginosa biofilms after exposure to Ciprofloxacin HCl (day 10 exposures). Ciprofloxacin Replicate #1 Replicate #2 Replicate #3 Average CFU/mL HCl (μg/mL) 10 0 0 0 0 5 0 0 0 0 2.5 5.6x105 8.0x105 7.2x105 6.9x105 0.5 4.7x105 5.6x105 4.3x105 4.9x105 Antimicrobial Activity of cave Bacterial Isolates on Planktonic P. aeruginosa The day 2, 4, 6, 8 and 10 samples were also used in a KB disc diffusion assay but none of the strains in any sample demonstrated any inhibition of P. aeruginosa on agar plates. Oddly even day 8 samples which exhibited antimicrobial effects against the biofilms, did not have an effect on planktonic P. aeruginosa. The day 8 samples were used in the KB disc diffusion assay 5 days after they were used in the Biofilm assay. When the samples were initially used, they were stored at -20oC, but after they were stored at 4oC, so it is possible that the higher temperature resulted in chemical changes to the molecules in the samples, rendering them ineffective against P. aeruginosa. However, an alternative idea is that the cave isolate supernatant inhibited the biofilm rather than killing the cells. Since the supernatant samples did not inhibit the planktonic cells, but did reduce the biofilms, it is possible that the cave isolate supernatants actually were able to disrupt the biofilms without killing the cells within. 28 The Antibiotic and Disinfectant Susceptibilities of P. aeruginosa in a Planktonic Form The same positive controls of 2% Virkon, tetracycline HCl, and ciprofloxacin (HCl) were used with the same concentrations as the biofilm assays. The 2% Virkon in day 2, 4, 6, 8, and 10 showed an inhibitory zone (too small to measure) around the discs. The diameter may have been small because of the chemical composition of Virkon. Virkon is composed of peroxygen compounds including Potassium peroxymonosulfate, which is a negatively charged particle, making it difficult to diffuse through the agar, which is also negatively charged. This idea is again consistent with that of Hernndez et al., (2000), which found that Virkon had bactericidal effects on P. aeruginosa. The antibiotic tetracycline HCl also showed inhibitory activity when used in the KB disc diffusion assay. For days 2, 4, 6, 8, and 10, tetracycline HCl at 120 μg/mL showed an inhibitory zone. Days 2, 4, and 8 showed measureable inhibitory zones that ranged from 10 to 14 mm. Also Days 4 and 8 showed inhibitory zones at 60 μg/mL, but were too small to measure. So the 60 μg/mL could be thought of as the MIC of tetracycline HCl on P. aeruginosa. This is within the range of other MIC’s found for P. aeruginosa (various lab strains such as PA01) which were 0.5-64 μg/mL (Li et al., 1994; Morita et al., 2001). The antibiotic ciprofloxacin (dissolved in 0.1 N HCl) was used for sample days 2, 4, 6, and 8 and ciprofloxacin HCl was used for sample day 10. Both showed very similar inhibitory activity when used in the KB disc diffusion assay. A control with just 0.1 N HCl was used for days 2, 4, 6, and 8 which showed that it had no inhibitory activity (a different result than the Biofilm assay mentioned earlier). For sample days 2, 4, 6, 8, and 10 the 29 ciprofloxacin/ciprofloxacin HCl showed inhibitory zone ranges of 26-19 mm and 18-10 mm for concentrations of 10 and 5 μg/mL respectively. Ciprofloxacin/ciprofloxacin HCl at 2.5 μg/mL showed an inhibitory zone of 12-10 mm for Day 2 then inhibitory zones too small to measure for Day 4, 6, 8, and 10. The concentration of 0.5 μg/mL had no inhibitory zone, indicating too low of a concentration to have an inhibitory effect on P. aeruginosa. This would indicate that 2.5 μg/mL is the MIC which is slightly higher than some other studies have found. Some studies put the MIC in a range of 0.1-0.5 μg/mL for various strains of P. aeruginosa including ATCC 27853 and PA01 (Ceri et al., 1999; Li et al., 1994; Morita et al., 2001), but a more recent study showed an MIC range of 0.25-2 μg/mL (Dosler and Karaaslan, 2014), which is a value much closer to what this study found. Antimicrobial Activity as Compared to Previous Studies with Same Isolates The results of this study correspond to earlier studies conducted with the cave isolates A1A3, RA003 and 58B. In a preliminary study, these cave isolates showed the ability to inhibit P. aeruginosa biofilms resulting in a reduced CFU/mL (Mason, 2015). The preliminary study found 58B V8 Non-UV as well as RA003 and A1A3 R2A Non-UV had inhibitory effects on biofilms. That study (Mason, 2015) and this one both show that biofilm inhibitory molecules are produced by the strains starting on day 7 (result of previous study) and day 8 (this study) but lose the activity by day 10 as shown in this study. The current study found that A1A3, RA003 and 58B could produce compounds that could inhibit P. aeruginosa biofilms. The cave isolate RA003 could produce antimicrobial compounds when cultured in R2A, ISP2 and V8 as well as being effective after the supernatant was exposed to UV light. This would suggest that RA003 is versatile organism that can grow in a variety of 30 culture media. In the preliminary study, RA003 was found to have inhibitory activity only in R2A. The cave isolate 58B was able to produce antimicrobial compounds when cultured in R2A and V8 as well. Previously in the preliminary study, 58B was only able to have inhibitory effects when cultured in V8. The cave isolate A1A3 was only able to produce antimicrobial compounds when cultured in V8. However in the preliminary study, it produced antimicrobial compounds in R2A (Mason, 2015). These cave isolates also have similar antimicrobial activities compared to previous studies conducted with these strains. The cave isolate A1A3 had antimicrobial effects against the Gram-negative bacteria Klebsiella pneumoniae, Escherichia coli and Acinetobacter baumannii having inhibitory zones of 11-13 mm when cultured in V8 (Sadoway, 2011) similar to this study. Also RA003 has shown activity against Micrococcus luteus and MDRMRSA when cultured in R2A, HT and ISP2, producing zones of inhibition 11-15mm (Alnahdi, 2014). This shows again that the cave isolate RA003 is able to produce antimicrobial compounds in a variety of media. Rule, (2012) found the cave isolate 58B had antimicrobial effects against Mycobacterium smegmatis and A. baumannii when cultured in HT. Sadoway, (2011) found that cave isolate 58B had antimicrobial effects on E. coli but only in HT and ISP2, not when cultured in V8 medium. Furthermore Alnahdi, 2014 found that the cave bacteria isolates cultured in R2A medium had high antimicrobial activities. Her study showed the 58B cave isolate cultured in R2A had antimicrobial effects against Micrococcus luteus, M. smegmatis, and Candida albicans. In addition, the RA003 cave isolate had antimicrobial activity against M. luteus when cultured in R2A medium. Alnahdi, (2014) identified R2A broth to be the optimal fermentation broth because the zones of inhibition for 58B and RA003 were larger when grown in R2A as compared to HT broth. 31 This study showed R2A was able to support isolates 58B and RA003 to make antimicrobial compounds, but it did not seem to be the most effective culture medium. The V8 culture medium was able to sustain A1A3, RA003 and 58B, which caused all three strains to produce compounds that could inhibit P. aeruginosa biofilms. The most interesting finding was that 58B cultured in the V8 medium produced significant reduction of the biofilms with and without UV treatment. In fact, the UV treated 58B V8 supernatant yielded a compound that in two replicates resulted in complete biofilm eradication, an impressive result considering an impure sample. Furthermore this study found that only one of the cave isolates showed antimicrobial activity after exposure to UV light for 30 minutes; however, it was the most effective sample. Sadoway, (2011) found that the cave isolate 58B only had an antimicrobial effect against E. coli when not exposed to UV. Also Rule, (2012) found that the same cave isolate again only had activity against M. smegmatis and A. baumannii when not exposed to UV light. In this study, it is possible that the UV light actually increased the antimicrobial compound’s antimicrobial effect by causing a conformation change. One study showed that the antibiotics oxytetracycline, doxycycline and ciprofloxacin had increased inhibition against Vibrio fisheri when exposed to 3816 mJ/cm2 of UV fluence (energy rate) (Yuan et al., 2011). Identities of Cave Bacterial Isolates and P. aeruginosa The clinically useful antibiotics used to treat P. aeruginosa are generally carbapenems, fluoroquinolones, and aminoglycosides (Morita et al., 2014). Since these cave bacterial isolates produced antimicrobial compounds that inhibited P. aeruginosa, it is possible that the compounds may resemble one of the previous antibiotics mentioned. In fact, 32 carbapenems and aminoglycosides have been known to be produced by Streptomyces species (Poole, 2011). The cave isolates A1A3 and 58B were shown to be Gram positive filamentous bacteria, which is consistent with Streptomyces species identification. Furthermore the 16S rRNA sequencing also showed that both A1A3 and 58B were Streptomyces species with 99% shared similarity and 100% query coverage (Table 3). However due to the similarity and query coverage, a species could not be discerned for either isolate. The cave isolate RA003 was a Gram positive rod shaped bacterium and the 16S rRNA sequencing agreed with this. The sequencing showed that the cave isolate RA003 was in the Bacillus genus but again due the similarity and query coverage a species could not be discerned (Table 3). The gene sequencing also showed that the pathogenic bacterium was P. aeruginosa but a strain could not be concluded based on the similarity and query coverage (Table 3). Members of the Bacillus genus has previously been able to produce bacteriocins (antimicrobial peptides) against a wide variety of bacteria. Some examples the bacterium’s inhibitory effects are: Listeria monocytogenes ATCC 19111, Pseudomonas fluorescens ATCC 11251, Staphylococcus aureus ATCC 25923 and E. coli (Abdel-Mohsein et al., 2011; He et al., 2006; Kayalvizhi and Gunasekaran, 2008). Table 3: Identification of the organisms used according to 16S rRNA gene sequencing Organism Possible Identity Percent Query Similarity Coverage Streptomyces fulvissimus strain DSM 40593 99 100 Streptomyces microflavus strain PM86A 99 100 Streptomyces fulvissimus strain DSM 40593 99 100 Streptomyces microflavus strain PM86A 99 100 Analyzed A1A3 58B 33 Bacillus pumilus strain Jo2 99 100 Bacillus altitudinis strain KUDC1731 99 100 Pseudomonas Pseudomonas aeruginosa strain SBTPe-001 99 100 aeruginosa Pseudomonas aeruginosa strain SV1 99 100 Pseudomonas aeruginosa strain VSS6 99 100 RA003 Microbial natural products are still a major source of new drugs for antibiotic production, but must be discovered first. Microorganisms from a wide variety of environments have been shown to be able to combat pathogenic bacteria and in some cases the biofilms they form. The biofilms are quite often a major cause of the pathogenicity of the bacterium in question. The bacterial strains used in this study were isolated from caves found in British Columbia, which further solidifies that natural habitats can produce many useful bacteria. Recently, a study used bacteria from soil, cave, and rivers to produce cell free extracts. The extracts were then used against Staphylococcus aureus biofilms in a 96 well microtitre plate, similar to this study. The study found that 55/126 extracts significantly inhibited the biofilms due a variety of chemical compounds found in the extracts. Also 40% of the extracts were found to contain DNase, which could break down extracellular DNA, a major structural component of some biofilms (Farmer et al., 2014). In another study, researchers collected bacterial samples from Magura Cave in Bulgaria. The samples were very diverse with respect to bacterial species, and all produced varying compounds such as proteases, xanthan lyase and β-glycosidase. Again the researchers found that 75% of the collected samples showed antimicrobial activity against Bacillus subtilis ATCC 6633 and Pseudomonas aeruginosa NBIMCC 1390 in a biofilm (Lazarkevich et al., 2013). Some 34 marine bacterial isolates have been studied as well for their biofilm effects. In one study bacterial strains from sediment in the Palk Bay region were isolated, then used to produce cell free extracts. These extracts did not show biofilm biocidal effects, but could inhibit the biofilms from forming. The marine isolates Bacillus indicus MTCC 5559 and Bacillus pumilus MTCC 5560 showed mature biofilms of P. aeruginosa being inhibited by 70-74% (Nithya et al., 2010). CONCLUSIONS AND FUTURE WORK Cave bacteria, including actinomycetes, continue to be important in the discovery of new bioactive compounds. The cave bacteria isolates A1A3, RA003, and 58B used in this project continued to have inhibitory activity against another pathogenic bacteria Pseudomonas aeruginosa. Of the 90 total supernatant samples collected from culturing for 2, 4, 6, 8 and 10 days (3 strains, 3 different media, 2 treatments, and 5 different days) only 8 of them showed inhibitory activity (approximately 9%) . These samples were all from the Day 8 collection, but only 2 of the samples showed significant inhibition of the P. aeruginosa biofilms. Since the samples had no effect on the planktonic cells, it is reasonable to think that there was a biofilm dispersal effect rather than a bactericidal effect. The fermentation broths of R2A and V8 seemed to be the most effective choices to produce secondary metabolites from cave bacteria. The V8 medium did yield antimicrobial secondary metabolites from all three strains used. The exposure of UV light did appear to have an activating effect on the samples RA003 and 58B both cultured in V8 and 58B turned out to be the more effective, having complete biofilm eradication properties. It is promising that these cave bacterial isolates produced secondary metabolites that would inhibit biofilms at all. Cells living in a 35 biofilm are more resistant to antimicrobial compounds, so it is impressive that the cave isolates could have inhibitory effects without any purification or concentrating. The next step would be to take samples from Day 7 to Day 9 in order to pinpoint where the most effective secondary metabolite it produced. The culture conditions such as temperature, pH, oxygen etc. could also be studied to optimize the antimicrobials produced. As well it would be important to purify and identify what specific molecules are present in the cave isolate samples in order to decipher the mechanism of action. It is obvious now that antibiotic susceptibilities of free-living bacteria are greatly reduced as compared to biofilm susceptibilities. The MIC of P. aeruginosa to the antibiotic ciprofloxacin HCl is lower (half according to this study) than what is need for the MBEC. Using the MBEC 96 well microtitre plate to study susceptibilities of biofilms to antimicrobials is essential. This high throughput method allows for more realistic conditions, giving more accurate susceptibilities of the organisms in their natural state. Plate counts were used in this study to evaluate the biofilm eradication activity of the cave isolates. This method is useful because it can distinguish between live and dead cells to give accurate cell concentration measurements. However, it is incredibly time intensive, and sometimes prone to contamination, which meant doing the trial over again. Using a plate reader would be much faster and more efficient than using plate counts. It would be best to use the plate reader for the initial biocidal activity, and then confirm the result with the plate counts. This would make the process more efficient while still retaining accurate results of whether the cells are alive or dead. As well this study used a laboratory strain of P. aeruginosa to evaluate the antimicrobial activity of the cave isolate samples. The next step may be to use 36 multi-drug resistant strains of P. aeruginosa from hospitals and determine if the cave bacterial isolates are still effective against those strains. Overall, the three cave bacteria isolates: A1A3, RA003, and 58B were able to have an inhibitory effect against P. aeruginosa biofilms. The Kirby-Bauer disc diffusion assay determined that the cave bacterial isolates did not have an effect on planktonic cells. The result of these two experiments would suggest that the cave isolates contain biofilm disrupting compounds. The scanning electron microscopy found that the bacteria did grow in a biofilm formation on the pegs of the MBEC device. 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Standard Test Method for Testing Disinfectant Efficiacy against Pseudomonas aeruginosa Biofilm using the MBEC Assay. 41 APPENDIX A-MEDIA Hickey-Tresner Media Yeast Extract 1.0 g Beef Extract 1.0 g N-Z Amine A 2.0 g Dextrin 10.0 g dH2O to 1 L pH 7.3 V8 Juice Media V8 supernatant 200.0 mL CaCO3 3.0 g dH2O 800.0 mL pH 6.0 *PC Blue Ribbon Low Sodium V8 juice centrifuged at 10,000 rpm for 10 minutes to collect supernatant *Media heated to dissolve Dextrin International Streptomyces Project # 2 (Yeast-Malt Extract) Yeast Extract 4.0 g Glucose 4.0 g Malt Extract 10.0 g dH2O to 1 L pH 7.3 R2A Media R2A pre-made broth media dH2O 3.15 g pH 7.0 to 1 L 42 D/E Neutralizing Broth Media Pre-made D/E Neutralizing broth media dH2O 34.0 g pH Trypticase Soy Broth Media 30.0 g to 1 L BBL pre-made Trypticase soy broth media dH2O 7.6 pH 7.3 to 1 L 43 APPENDIX B-RAW DATA Day 2 Biofilm recovery growth check (+=Growth, -= No growth, RED= reduced growth) Non #1 A1A3 R2A NON + A1A3 ISP2 NON A1A3 V8 NON R003 R2A NON + + + #2 + + + #3 + + UV #1 A1A3 R2A + A1A3 ISP2 + #2 + #3 Antibioti c R003 ISP2 NON R003 V8 NON 58B R2A NON 58B ISP2 NON 58B V8NON + + + + + + + + + + + + + + + + + + A1A3 V8 R003 ISP2 + R003 V8 58B R2A 58B ISP2 58B V8 + R003 R2A + + + + + + + + + + + + + + + + + + + + + + 60 30 10 Cipro 10 ug/mL 5 2.5 0.5 #1 Tetra HCl 120 ug/mL + + + + - - - - #2 + + + + - - - - #3 + + + + - - - - R2A sterile V8 sterile control - Sterile TSB 2% Virkon 0.1 N HCl + - - 1# - ISP2 sterile control - #2 - - - + - - #3 - - - + - - Day 4 Biofilm recovery growth check (+=Growth, -= No growth, RED= reduced growth) Non A1A3 A1A3 A1A3 V8 R003 R003 R003 V8 58B R2A 58B ISP2 58B 44 #1 R2A NON + ISP2 NON NON R2A NON ISP2 NON NON + + + + + + + + #2 + + + + + + + + + #3 + + + + + + + + + UV A1A3 ISP2 + A1A3 V8 R003 ISP2 + 58B R2A 58B ISP2 58B V8 + R003 R2A + R003 V8 #1 A1A3 R2A + + + + + #2 + + + + + + + + + #3 + + + + + + + + + Antibioti c 60 30 10 Cipro 10 ug/mL 5 2.5 0.5 #1 Tetra HCl 120 ug/mL + + + + - - - - #2 + + + + - - - - #3 + + + + - - - - R2A sterile V8 sterile control - Sterile TSB 2% Virkon 0.1 N HCl + - - 1# - ISP2 sterile control - #2 - - - + - - #3 - - - + - - NON NON V8NON Day 6 Biofilm recovery growth check (+=Growth, -= No growth, RED= reduced growth) Non #1 A1A3 R2A NON + A1A3 ISP2 NON A1A3 V8 NON R003 R2A NON + + + #2 + + + #3 + + + R003 ISP2 NON R003 V8 NON 58B R2A NON 58B ISP2 NON 58B V8NON + + + + + + + + + + + + + + + + + 45 UV #1 A1A3 R2A + A1A3 ISP2 + #2 + #3 Antibioti c A1A3 V8 + R003 R2A + R003 ISP2 + R003 V8 58B R2A 58B ISP2 58B V8 + + + + + + + + + + + + + + + + + + + + + 60 30 #1 Tetra HCl 120 ug/mL + 10 Cipro 10 ug/mL 5 2.5 0.5 + + + - - - - #2 + + + + - - - - #3 + + + + - - - - R2A sterile V8 sterile control - Sterile TSB 2% Virkon 0.1 N HCl + - - 1# - ISP2 sterile control - #2 - - - + - - #3 - - - + - - Day 8 Biofilm recovery growth check (+=Growth, -= No growth, RED= reduced growth) Non A1A3 ISP2 NON + A1A3 V8 NON #1 A1A3 R2A NON + R003 ISP2 NON RED R003 V8 NON 58B R2A NON 58B ISP2 NON 58B V8NON RED R003 R2A NON RED RED - + RED #2 + + + + + + + + + #3 + + + + + RED + + + UV A1A3 ISP2 + R003 ISP2 + 58B R2A 58B ISP2 58B V8 + R003 R2A + R003 V8 #1 A1A3 R2A + + + + + #2 + + + + + + + + - A1A3 V8 46 RED + + - 10 Cipro 10 ug/mL 5 2.5 0.5 + + - - - - + + + - - - - + + + + - - - - R2A sterile V8 sterile control - Sterile TSB 2% Virkon 0.1 N HCl + - - #3 + + + + Antibioti c 60 30 #1 Tetra HCl 120 ug/mL + + #2 + #3 + 1# - ISP2 sterile control - #2 - - - + - - #3 - - - + - - Day 10 Biofilm recovery growth check (+=Growth, -= No growth, RED= reduced growth) Non #1 A1A3 R2A NON + A1A3 ISP2 NON A1A3 V8 NON R003 R2A NON + + + #2 + + + #3 + + UV #1 A1A3 R2A + A1A3 ISP2 + #2 + #3 Antibioti c R003 ISP2 NON R003 V8 NON 58B R2A NON 58B ISP2 NON 58B V8NON + + + + + + + + + + + + + + + + + + A1A3 V8 R003 ISP2 + R003 V8 58B R2A 58B ISP2 58B V8 + R003 R2A + + + + + + + + + + + + + + + + + + + + + + Tetra HCl 120 ug/mL 60 30 10 Cipro 10 ug/mL 5 2.5 0.5 47 #1 + + + + - - + + #2 + + + + - - + + #3 + + + + - - + + R2A sterile V8 sterile control - Sterile TSB 2% Virkon sterile water + - + 1# - ISP2 sterile control - #2 - - - + - + #3 - - - + - + Day 2 KB disc diffusion( +=Inhibition, -= NO inhibition, #= diameter of inhibitory zone in mm) Non A1A3 ISP2 NON - A1A3 V8 NON #1 A1A3 R2A NON - R003 ISP2 NON - R003 V8 NON 58B R2A NON 58B ISP2 NON 58B V8NON - R003 R2A NON - - - - - #2 - - - - - - - - - #3 - - - - - - - - - UV A1A3 ISP2 - R003 ISP2 - 58B R2A 58B ISP2 58B V8 - R003 R2A - R003 V8 #1 A1A3 R2A - - - - - #2 - - - - - - - - - #3 - - - - - - - - - Antibioti c 60 30 10 Cipro 10 ug/mL 5 2.5 0.5 #1 Tetra HCl 120 ug/mL 10 - - - 26 18 12 - #2 + - - - 25 16 10 - A1A3 V8 48 #3 + - - - 24 R2A sterile V8 sterile control - Sterile TSB 2% Virkon 0.1 N HCl - + - 1# - ISP2 sterile control - #2 - - - - + - #3 - - - - + - 18 10 - Day 4 KB disc diffusion( +=Inhibition, -= NO inhibition, #= diameter of inhibitory zone in mm) Non A1A3 ISP2 NON - A1A3 V8 NON #1 A1A3 R2A NON - R003 ISP2 NON - R003 V8 NON 58B R2A NON 58B ISP2 NON 58B V8NON - R003 R2A NON - - - - - #2 - - - - - - - - - #3 - - - - - - - - - UV A1A3 ISP2 - R003 ISP2 - 58B R2A 58B ISP2 58B V8 - R003 R2A - R003 V8 #1 A1A3 R2A - - - - - #2 - - - - - - - - - #3 - - - - - - - - - Antibioti c 60 30 10 Cipro 10 ug/mL 5 2.5 0.5 #1 Tetra HCl 120 ug/mL 14 - - - 23 15 + - #2 14 - - - 21 17 + - #3 13 - - - 20 15 + - R2A sterile ISP2 sterile V8 sterile Sterile TSB A1A3 V8 2% Virkon 0.1 N HCl 49 control control 1# - - - - + - #2 - - - - + - #3 - - - - + - Day 6 KB disc diffusion( +=Inhibition, -= NO inhibition, #= diameter of inhibitory zone in mm) Non A1A3 ISP2 NON - A1A3 V8 NON #1 A1A3 R2A NON - R003 ISP2 NON - R003 V8 NON 58B R2A NON 58B ISP2 NON 58B V8NON - R003 R2A NON - - - - - #2 - - - - - - - - - #3 - - - - - - - - - UV A1A3 ISP2 - R003 ISP2 - 58B R2A 58B ISP2 58B V8 - R003 R2A - R003 V8 #1 A1A3 R2A - - - - - #2 - - - - - - - - - #3 - - - - - - - - - Antibioti c 60 30 10 Cipro 10 ug/mL 5 2.5 0.5 #1 Tetra HCl 120 ug/mL + - - - 23 16 + - #2 + - - - 20 15 + - #3 + - - - 21 12 + - R2A sterile ISP2 sterile control V8 sterile control Sterile TSB A1A3 V8 2% Virkon 0.1 N HCl 50 1# - - - - + - #2 - - - - + - #3 - - - - + - Day 8 KB disc diffusion ( +=Inhibition, -= NO inhibition, #= diameter of inhibitory zone in mm) Non A1A3 ISP2 NON - A1A3 V8 NON #1 A1A3 R2A NON - R003 ISP2 NON - R003 V8 NON 58B R2A NON 58B ISP2 NON 58B V8NON - R003 R2A NON - - - - - #2 - - - - - - - - - #3 - - - - - - - - - UV A1A3 ISP2 - R003 ISP2 - 58B R2A 58B ISP2 58B V8 - R003 R2A - R003 V8 #1 A1A3 R2A - - - - - #2 - - - - - - - - - #3 - - - - - - - - - Antibioti c 60 30 10 Cipro 10 ug/mL 5 2.5 0.5 #1 Tetra HCl 120 ug/mL 10 + - - 20 14 + - #2 12 + - - 20 11 + - #3 11 + - - 21 12 + - R2A sterile ISP2 sterile control - V8 sterile control - Sterile TSB 2% Virkon 0.1 N HCl - + - 1# - A1A3 V8 51 #2 - - - - + - #3 - - - - + - Day 10 KB disc diffusion (+=Inhibition, -= NO inhibition, #= diameter of inhibitory zone in mm) Non A1A3 ISP2 NON - A1A3 V8 NON - R003 R2A NON - R003 ISP2 NON - R003 V8 NON - 58B R2A NON - 58B ISP2 NON - 58B V8NON #1 A1A3 R2A NON - #2 - - - - - - - - - #3 - - - - - - - - - UV A1A3 ISP2 - A1A3 V8 - R003 R2A - R003 ISP2 - R003 V8 - 58B R2A - 58B ISP2 - 58B V8 #1 A1A3 R2A - #2 - - - - - - - - - #3 - - - - - - - - - Antibio tic 60 30 10 Cipro 10 ug/mL 5 2.5 0.5 #1 Tetra HCl 120 ug/mL + + - - 19 11 + - #2 + - - - 19 10 + - #3 + - - - 20 11 + - R2A sterile ISP2 sterile control - V8 sterile control - Sterile TSB 2% Virkon - + 1# - - - Water sterile control - 52 #2 - - - - + - #3 - - - - + - 53 APPENDIX C-CONTIGUOUS SEQUENCES OF BACTERIA > Contig - A1A3_518-800 GGACGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAACGATGAAGCCTTTCGGGGTGGATTAGTGGC GAACGGGTGAGTAACACGTGGGCAATCTGCCCTTCACTCTGGGACAAGCCCTGGAAACGGGGTCTAATAC CGGATAACACTCTGTCCTGCATGGGACGGGGTTAAAAGCTCCGGCGGTGAAGGATGAGCCCGCGGCCTAT CAGCTTGTTGGTGGGGTAATGGCCTACCAAGGCGACGACGGGTAGCCGGCCTGAGAGGGCGACCGGCCAC ACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGAAAG CCTGATGCAGCGACGCCGCGTGAGGGATGACGGCCTTCGGGTTGTAAACCTCTTTCAGCAGGGAAGAAGC GAAAGTGACGGTACCTGCAGAAGAAGCGCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGCG CAAGCGTTGTCCGGAATTATTGGGCGTAAAGAGCTCGTAGGCGGCTTGTCACGTCGGATGTGAAAGCCCG GGGCTTAACCCCGGGTCTGCATTCGATACGGGCTAGCTAGAGTGTGGTAGGGGAGATCGGAATTCCTGGT GTAGCGGTGAAATGCGCAGATATCAGGAGGAACACCGGTGGCGAAGGCGGATCTCTGGGCCATTACTGAC GCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGTTGGGA ACTAGGTGTTGGCGACATTCCACGTCGTCGGTGCCGCAGCTAACGCATTAAGTTCCCCGCCTGGGGAGTA CGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCAGCGGAGCATGTGGCTTAATTC GACGCAACGCGAAGAACCTTACCAAGGCTTGACATATACCGGAAAGCATCAGAGATGGTGCCCCCCTTGT GGTCGGTATACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACG AGCGCAACCCTTGTTCTGTGTTGCCAGCATGCCCTTCGGGGTGATGGGGACTCACAGGAGACTGCCGGGG TCAACTCGGAGGAAGGTGGGGACGACGTCAAGTCATCATGCCCCTTATGTCTTGGGCTGCACACGTGCTA CAATGGCCGGTACAATGAGCTGCGATGCCGCGAGGCGGAGCGAATCTCAAAAAGCCGGTCTCAGTTCGGA TTGGGGTCTGCAACTCGACCCCATGAAGTCGGAGTTGCTAGTAATCGCAGATCAGCATTGCTGCGGTGAA TACGTTCCCGGGCCTTGTACACACCGCCCGTCACGTCACGAAAGTCGGTAACACCCGAAGCCGGTGGCCC AACCCCTTGTGGGAGGGAGCTGTCGAAGGTGGGACTGGCGATTG > Contig - A1A3_1492-27 GTTGGGCCACCGGCTTCAGGTGTTACCGACTTTCGTGACGTGACGGGCGGTGTGTACAAGGCCCGGGAAC GTATTCACCGCAGCAATGCTGATCTGCGATTACTAGCAACTCCGACTTCATGGGGTCGAGTTGCAGACCC CAATCCGAACTGAGACCGGCTTTTTGAGATTCGCTCCGCCTCGCGGCATCGCAGCTCATTGTACCGGCCA TTGTAGCACGTGTGCAGCCCAAGACATAAGGGGCATGATGACTTGACGTCGTCCCCACCTTCCTCCGAGT TGACCCCGGCAGTCTCCTGTGAGTCCCCATCACCCCGAAGGGCATGCTGGCAACACAGAACAAGGGTTGC GCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAGCCATGCACCACCTGTATACCG ACCACAAGGGGGGCACCATCTCTGATGCTTTCCGGTATATGTCAAGCCTTGGTAAGGTTCTTCGCGTTGC GTCGAATTAAGCCACATGCTCCGCTGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTTAGCCTTGCGG CCGTACTCCCCAGGCGGGGAACTTAATGCGTTAGCTGCGGCACCGACGACGTGGAATGTCGCCAACACCT AGTTCCCAACGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCTCCTC AGCGTCAGTAATGGCCCAGAGATCCGCCTTCGCCACCGGTGTTCCTCCTGATATCTGCGCATTTCACCGC TACACCAGGAATTCCGATCTCCCCTACCACACTCTAGCTAGCCCGTATCGAATGCAGACCCGGGGTTAAG CCCCGGGCTTTCACATCCGACGTGACAAGCCGCCTACGAGCTCTTTACGCCCAATAATTCCGGACAACGC TTGCGCCCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGGCGCTTCTTCTGCAGGTACCGTCACT TTCGCTTCTTCCCTGCTGAAAGAGGTTTACAACCCGAAGGCCGTCATCCCTCACGCGGCGTCGCTGCATC AGGCTTTCGCCCATTGTGCAATATTCCCCACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCC AGTGTGGCCGGTCGCCCTCTCAGGCCGGCTACCCGTCGTCGCCTTGGTAGGCCATTACCCCACCAACAAG CTGATAGGCCGCGGGCTCATCCTTCACCGCCGGAGCTTTTAACCCCGTCCCATGCAGGACAGAGTGTTAT CCGGTATTAGACCCCGTTTCCAGGGCTTGTCCCAGAGTGAAGGGCAGATTGCCCACGTGTTACTCACCCG TTCGCCACTAATCCACCCCGAAAGGATTCAT 54 > Contig - PAB_518-800 AGATTGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAGCGGATGAAGGGAGCTTGCTCCTGGATTCA GCGGCGGACGGGTGAGTAATGCCTAGGAATCTGCCTGGTAGTGGGGGATAACGTCCGGAAACGGGCGCTA ATACCGCATACGTCCTGAGGGAGAAAGTGGGGGATCTTCGGACCTCACGCTATCAGATGAGCCTAGGTCG GATTAGCTAGTTGGTGGGGTAAAGGCCTACCAAGGCGACGATCCGTAACTGGTCTGAGAGGATGATCAGT CACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGA AAGCCTGATCCAGCCATGCCGCGTGTGTGAAGAAGGTCTTCGGATTGTAAAGCACTTTAAGTTGGGAGGA AGGGCAGTAAGTTAATACCTTGCTGTTTTGACGTTACCAACAGAATAAGCACCGGCTAACTTCGTGCCAG CAGCCGCGGTAATACGAAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCGCGTAGGTGGTTC AGCAAGTTGGATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATCCAAAACTACTGAGCTAGAGTACGG TAGAGGGTGGTGGAATTTCCTGTGTAGCGGTGAAATGCGTAGATATAGGAAGGAACACCAGTGGCGAAGG CGACCACCTGGACTGATACTGACACTGAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGT AGTCCACGCCGTAAACGATGTCGACTAGCCGTTGGGATCCTTGAGATCTTAGTGGCGCAGCTAACGCGAT AAGTCGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCG GTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCTGGCCTTGACATGCTGAGAACTTTCC AGAGATGGATTGGTGCCTTCGGGAACTCAGACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAG ATGTTGGGTTAAGTCCCGTAACGAGCGCAACCCTTGTCCTTAGTTACCAGCACCTCGGGTGGGCACTCTA AGGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGGCCAGG GCTACACACGTGCTACAATGGTCGGTACAAAGGGTTGCCAAGCCGCGAGGTGGAGCTAATCCCATAAAAC CGATCGTAGTCCGGATCGCAGTCTGCAACTCGACTGCGTGAAGTCGGAATCGCTAGTAATCGTGAATCAG AATGTCACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGGAGTGGGTTGCTCC AGAAGTAGCTAGTCTAACCGCAAGGGGGACGGTTACCACGGAGTGATCATGATGGGTG > Contig - PAB_1492-27 GACTTGCTACTTCTGGAGCAACCCACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTAT TCACCGTGACATTCTGATTCACGATTACTAGCGATTCCGACTTCACGCAGTCGAGTTGCAGACTGCGATC CGGACTACGATCGGTTTTATGGGATTAGCTCCACCTCGCGGCTTGGCAACCCTTTGTACCGACCATTGTA GCACGTGTGTAGCCCTGGCCGTAAGGGCCATGATGACTTGACGTCATCCCCACCTTCCTCCGGTTTGTCA CCGGCAGTCTCCTTAGAGTGCCCACCCGAGGTGCTGGTAACTAAGGACAAGGGTTGCGCTCGTTACGGGA CTTAACCCAACATCTCACGACACGAGCTGACGACAGCCATGCAGCACCTGTGTCTGAGTTCCCGAAGGCA CCAATCCATCTCTGGAAAGTTCTCAGCATGTCAAGGCCAGGTAAGGTTCTTCGCGTTGCTTCGAATTAAA CCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCATTTGAGTTTTAACCTTGCGGCCGTACTCCCC AGGCGGTCGACTTATCGCGTTAGCTGCGCCACTAAGATCTCAAGGATCCCAACGGCTAGTCGACATCGTT TACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCACCTCAGTGTCAGTATCA GTCCAGGTGGTCGCCTTCGCCACTGGTGTTCCTTCCTATATCTACGCATTTCACCGCTACACAGGAAATT CCACCACCCTCTACCGTACTCTAGCTCAGTAGTTTTGGATGCAGTTCCCAGGTTGAGCCCGGGGATTTCA CATCCAACTTGCTGAACCACCTACGCGCGCTTTACGCCCAGTAATTCCGATTAACGCTTGCACCCTTCGT ATTACCGCGGCTGCTGGCACGAAGTTAGCCGGTGCTTATTCTGTTGGTAACGTCAAAACAGCAAGGTATT AACTTACTGCCCTTCCTCCCAACTTAAAGTGCTTTACAATCCGAAGACCTTCTTCACACACGCGGCATGG CTGGATCAGGCTTTCGCCCATTGTCCAATATTCCCCACTGCTGCCTCCCGTAGGAGTCTGGACCGTGTCT CAGTTCCAGTGTGACTGATCATCCTCTCAGACCAGTTACGGATCGTCGCCTTGGTAGGCCTTTACCCCAC CAACTAGCTAATCCGACCTAGGCTCATCTGATAGCGTGAGGTCCGAAGATCCCCCACTTTCTCCCTCAGG ACGTATGCGGTATTAGCGCCCGTTTCCGGACGTTATCCCCCACTACCAGGCAGATTCCTAGGCATTACTC ACCCGTCCGCCGCTGAATCCAGGAGCAAGCTCCCT 55 > Contig - PM58B_518-800 AGGACGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAACGATGAAGCCTTTCGGGGTGGATTAGTGG CGAACGGGTGAGTAACACGTGGGCAATCTGCCCTTCACTCTGGGACAAGCCCTGGAAACGGGGTCTAATA CCGGATAACACTCTGTCCTGCATGGGACGGGGTTAAAAGCTCCGGCGGTGAAGGATGAGCCCGCGGCCTA TCAGCTTGTTGGTGGGGTAATGGCCTACCAAGGCGACGACGGGTAGCCGGCCTGAGAGGGCGACCGGCCA CACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGAAA GCCTGATGCAGCGACGCCGCGTGAGGGATGACGGCCTTCGGGTTGTAAACCTCTTTCAGCAGGGAAGAAG CGAAAGTGACGGTACCTGCAGAAGAAGCGCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGC GCAAGCGTTGTCCGGAATTATTGGGCGTAAAGAGCTCGTAGGCGGCTTGTCACGTCGGATGTGAAAGCCC GGGGCTTAACCCCGGGTCTGCATTCGATACGGGCTAGCTAGAGTGTGGTAGGGGAGATCGGAATTCCTGG TGTAGCGGTGAAATGCGCAGATATCAGGAGGAACACCGGTGGCGAAGGCGGATCTCTGGGCCATTACTGA CGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGTTGGG AACTAGGTGTTGGCGACATTCCACGTCGTCGGTGCCGCAGCTAACGCATTAAGTTCCCCGCCTGGGGAGT ACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCAGCGGAGCATGTGGCTTAATT CGACGCAACGCGAAGAACCTTACCAAGGCTTGACATATACCGGAAAGCATCAGAGATGGTGCCCCCCTTG TGGTCGGTATACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAAC GAGCGCAACCCTTGTTCTGTGTTGCCAGCATGCCCTTCGGGGTGATGGGGACTCACAGGAGACTGCCGGG GTCAACTCGGAGGAAGGTGGGGACGACGTCAAGTCATCATGCCCCTTATGTCTTGGGCTGCACACGTGCT ACAATGGCCGGTACAATGAGCTGCGATGCCGCGAGGCGGAGCGAATCTCAAAAAGCCGGTCTCAGTTCGG ATTGGGGTCTGCAACTCGACCCCATGAAGTCGGAGTTGCTAGTAATCGCAGATCAGCATTGCTGCGGTGA ATACGTTCCCGGGCCTTGTACACACCGCCCGTCACGTCACGAAAGTCGGTAACACCCGAAGCCGGTGGCC CAACCCCTTGTGGGAGGGAGCTGTCGAAGGTGGGACTGGCGA > Contig - PM58B_1492-27 CGACTTTCGTGACGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCAGCAATGCTGATCTG CGATTACTAGCAACTCCGACTTCATGGGGTCGAGTTGCAGACCCCAATCCGAACTGAGACCGGCTTTTTG AGATTCGCTCCGCCTCGCGGCATCGCAGCTCATTGTACCGGCCATTGTAGCACGTGTGCAGCCCAAGACA TAAGGGGCATGATGACTTGACGTCGTCCCCCACCTTCCTCCGAGTTGACCCCGGCAGTCTCCTGTGAGTC CCCATCACCCCGAAGGGCATGCTGGCAACACAGAACAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACA TCTCACGACACGAGCTGACGACAGCCATGCACCACCTGTATACCGACCACAAGGGGGGCACCATCTCTGA TGCTTTCCGGTATATGTCAAGCCTTGGTAAGGTTCTTCGCGTTGCGTCGAATTAAGCCACATGCTCCGCT GCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTTAGCCTTGCGGCCGTACTCCCCAGGCGGGGAACTTA ATGCGTTAGCTGCGGCACCGACGACGTGGAATGTCGCCAACACCTAGTTCCCAACGTTTACGGCGTGGAC TACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCTCCTCAGCGTCAGTAATGGCCCAGAGATCC GCCTTCGCCACCGGTGTTCCTCCTGATATCTGCGCATTTCACCGCTACACCAGGAATTCCGATCTCCCCT ACCACACTCTAGCTAGCCCGTATCGAATGCAGACCCGGGGTTAAGCCCCGGGCTTTCACATCCGACGTGA CAAGCCGCCTACGAGCTCTTTACGCCCAATAATTCCGGACAACGCTTGCGCCCTACGTATTACCGCGGCT GCTGGCACGTAGTTAGCCGGCGCTTCTTCTGCAGGTACCGTCACTTTCGCTTCTTCCCTGCTGAAAGAGG TTTACAACCCCGAAGGCCGTCATCCCTCACGCGGGCGTCGCTGCATCAGGGCTTTCGCCCATTGTGCAAT ATTCCCCACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGGTCGCCCTCTCA GGCCGGCTACCCGTCGTCGCCTTGGTAGGCCATTACCCCACCAACAAGCTGATAGGCCGCGGGCTCATCC TTCACCGCCGGAGCTTTTAACCCCGTCCCATGCAGGACAGAGTGTTATCCGGTATTAGACCCCGTTTCCA GGGCTTGTCCCAGAGTGAAGGGCAGATTGCCCACGTGTTACTCACCCGTTCGCCACTAATCCACCCCGAA AGGATTCATCGTC 56 > Contig - RA003_518-800 AGGACGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAGCGAACAGAAGGGAGCTTGCTCCCGGATGT TAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGAG CTAATACCGGATAGTTCCTTGAACCGCATGGTTCAAGGATGAAAGACGGTTTCGGCTGTCACTTACAGAT GGACCCGCGGCGCATTAGCTAGTTGGTGGGGTAATGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAG AGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTC CGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGT TGTTAGGGAAGAACAAGTGCGAGAGTAACTGCTCGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTA ACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCT CGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGAA ACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACAC CAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATT AGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTG CAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGG GCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATC CTCTGACAACCCTAGAGATAGGGCTTTCCCTTCGGGGACAGAGTGACAGGTGGTGCATGGTTGTCGTCAG CTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTTAG TTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCC CTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCTGCGAGACCGCAAGGTTTAGCCA ATCCCATAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTA ATCGCGGATCAGCATGCCCGGGGGAATACGTTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGA GTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTATGGAGCCAGCCGCCGAAGGTGGGACAGATGATTGG GGTGAATCTAAGGGG > Contig - RA003_1492-27 ACTCTCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGA TTACTAGCGATTCCAGCTTCACGCAGTCGAGTTGCAGACTGCGATCCGAACTGAGAACAGATTTATGGGA TTGGCTAAACCTTGCGGTCTCGCAGCCCTTTGTTCTGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAA GGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCACCTTTAGAGTGCCC AACTAAATGCTGGCAACTAAGATCAAGGGTTGCGCTCGTTGCGGGACTTAACCCCAACATCTCACGACAC GAGCTGACGACAACCATGCACCACCTGTCACTCTGTCCCCGAAGGGAAAGCCCTATCTCTAGGGTTGTCA GAGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCG GGCCCCCGTCAATTCCTTTGAGTTTCAGTCTTGCGACCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAG CTGCAGCACTAAGGGGCGGAAACCCCCTAACACTTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTA TCTAATCCTGTTCGCTCCCCACGCTTTCGCTCCTCAGCGTCAGTTACAGACCAGAGAGTCGCCTTCGCCA CTGGTGTTCCTCCACATCTCTACGCATTTCACCGCTACACGTGGAATTCCACTCTCCTCTTCTGCACTCA AGTTTCCCAGTTTCCAATGACCCTCCCCGGTTGAGCCGGGGGCTTTCACATCAGACTTAAGAAACCGCCT GCGAGCCCTTTACGCCCAATAATTCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGT AGTTAGCCGTGGCTTTCTGGTTAGGTACCGTCAAGGTGCGAGCAGTTACTCTCGCACTTGTTCTTCCCTA ACAACAGAGCTTTACGATCCGAAAACCTTCATCACTCACGCGGCGTTGCTCCGTCAGACTTTCGTCCATT GCGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCAC CCTCTCAGGTCGGCTACGCATCGTCGCCTTGGTGAGCCATTACCCCACCAACTAGCTAATGCGCCGCGGG TCCATCTGTAAGTGACAGCCGAAACCGTCTTTCATCCTTGAACCATGCGGTTCAAGGAACTATCCGGTAT TAGCTCCGGTTTCCCGGAGTTATCCCAGTCTTACAGGCAGGTTACCCACGTGTTACTCACCCGTCCGCCG CTAACATCCGGGAGC 57