Faculty of Science VIRAL WARRIORS: SCREENING WATER SAMPLES FOR BACTERIOPHAGES AGAINST MULTIPLE DRUG RESISTANT BACTERIA 2016 | EMMA ELEANOR BEATRICE PERSAD B.Sc. Honours thesis VIRAL WARRIORS: SCREENING WATER SAMPLES FOR BACTERIOPHAGES AGAINST MULTIPLE DRUG RESISTANT BACTERIA by EMMA ELEANOR BEATRICE PERSAD 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, & Microbial Biology) This thesis has been accepted as conforming to the required standards by: Naowarat (Ann) Cheeptham (Ph.D.), Thesis Supervisor, Dept. Biological Sciences Joanna Urban (M.Sc., Ph.D. candidate), Co-supervisor, Dept. Biological Sciences Ken Wagner (M.D., FRCP), Co-supervisor, Dept. Biological Sciences Kingsley Donkor (Ph.D.), Examining Committee member, Dept. Chemistry Dated this 13th day of April, 2016, in Kamloops, British Columbia, Canada © Emma Eleanor Beatrice Persad, 2016 ABSTRACT Although mutation occurs randomly in nature and is passed randomly between bacterial species, the misuse and overuse of antibiotics in modern medicine has selected for antibiotic resistant organisms, resulting in an epidemic of antibiotic resistant infections. Used extensively in former Soviet Union countries with success, Western researchers have begun considering phage therapy for treatments, however it must be subjected to rigorous clinical trials before it can be approved by the FDA as a treatment method in North America (Gill et al. 2010; Abhilash et al. 2009). In this study, phage screening was performed on eight MDR bacterial strains provided from LifeLabs and Royal Inland Hospital in Kamloops, B.C.: E. coli 15-102, 15-124, and 14-318; Micrococcus luteus; Methicillin Resistant Staphylococcus Aureus (MRSA) 1 and 2; Serratia marcescens; and Mycobacterium smegmatis. One non-resistant E. coli strain known to be killed by phages found in Kamloops sewage was used as a positive control. Seven water samples and one non-water sample were used in this experiment as a source of phages. Water samples were obtained from the Kamloops Sewage Treatment Plant, the Domtar pulp mill run-off, the Pacific Ocean, Bisaro Anima Cave, and alkaline ponds around Kamloops. The non-water sample was created from mixing dirt from Abbotsford, B.C. with sterile water. An additional enriched water sample was made through the incubation of broth culture, nutrient broth, and sewage water overnight at 37°C in an attempt to select for more strain-specific phages (Prescott et al. 2005). In addition, sterile water was used in the protocol as a negative control. The successfulness of each phage screening trial was measured through the formation of plaques, which developed after plating the Multiple Drug Resistant (MDR) bacteria, molten agar, and phages for confluent growth on nutrient agar (Prescott et al. 2005). Of all the bacteria and environmental water samples, plaques only developed for the E. coli 14-318 strain using sewage water from the Kamloops Sewage Treatment Center. Using phage screening against these MDR bacteria allowed us to see that MDR pathogens present in our community are treatable with a potentially more beneficial and successful method to antibiotics. Thesis Supervisor: Dr. Naowarat Cheeptham ii ACKNOWLEDGEMENTS I would like to express my gratitude to my research professor Naowarat Cheeptham for her continuous support, knowledge, and patience displayed throughout this project. I would also like to thank my co-supervisors, Joanna Urban and Dr. Ken Wagner, for their insightful input and encouragement, along with lab technician, May Al-Fouadi, for her support and expertise in the laboratory. In addition, I would like to thank Dr. Kelly and Wendy Cummer at LifeLabs for the bacteria strains, Kathleen Graham for providing the cave water samples, Domtar, the Kamloops Sewage Treatment Plant, and Louis Gosselin for the seawater sample. Furthermore, I would also like to thank our collaborator Dr. Thomas Smith from the University of Texas, fellow lab partner Cindy Lam, and Kingsley Donkor, who is a member of my thesis committee. Finally, I would like to thank the research office for awarding me an Undergraduate Research Experience Award Program Scholarship. iii TABLE OF CONTENTS ABSTRACT.................................................................................................................................... ii ACKNOWLEDGEMENTS ............................................................................................................ ii TABLE OF CONTENTS ............................................................................................................... iv LIST OF FIGURES ........................................................................................................................ v LIST OF TABLES ......................................................................................................................... vi INTRODUCTION ......................................................................................................................... vi MATERIALS AND METHODS .................................................................................................. 10 RESULTS ..................................................................................................................................... 12 DISCUSSION ............................................................................................................................... 19 LITERATURE CITED ................................................................................................................. 23 iv LIST OF FIGURES Figure 1. The lytic, lysogenic, and pseudolysogenic cycles. a. The lytic bacteriophage cycle, in which the phage replicates using host machinery and lyses the cell; b. The lysogenic cycle, where the phage genome is incorporated into the host chromosome as a prophage and persists in a dormant state until environmental stressors trigger the lytic cycle to commence; c. Pseudolysogeny, in which phage genome fails to replicate or establish itself as a prophage, occurring typically in nutrient-deprived conditions (Feiner et al. 2015). Figure 2. Phage induced bacteriolysis: (1) Phage adsorption and DNA injection; (2) Phage DNA replication; (3) head and tail production; (4) holin and lysin synthesis; (5) DNA packaging; (6) complete phage assembly; (7) lysis of cell wall and release of phages; (8) circularization of phage DNA; (9) integration of phage DNA into host genome via lytic cycle (Matsuzaki et al. 2005). Figure 3. Overview of experimental procedure outlining the isolation and purification of bacteriophages from water samples, growth of bacteria in nutrient broth, and combination of phage water, bacteria, and molten agar on nutrient agar plate. Figure 4. Plaque presence after plating bacterial strain E. coli 14-318 with molten agar and sewage water and sewage broth samples in different concentrations: 1. 0.5 mL sewage water to 0.5 mL bacterial culture; 2. 0.5 mL sewage broth to 0.5 mL bacterial culture; 3. 0.9 mL sewage water to 0.1 mL bacterial culture; and 4. 0.9 mL sewage broth to 0.1 mL bacterial culture. v LIST OF TABLES Table 1. The resistance, possession of carbapenemase and ESBL, and plaque presence observed of the MDR bacteria used in this project. Table 2. The water sample location and presence of plaques when water samples had been plated with MDR bacteria and molten agar. Table 3. The different water sample (enriched or regular sewage) to bacterial broth dilutions and the number and size of plaques present. Table 4. Additional resistance of E. coli 14-318 to beta lactams, aminoglycosides, quinolones, tetracyclines, furanes, and trimethoprim/sulfonamides. vi vii INTRODUCTION BACKGROUND AND RATIONALE Importance of Phage Therapy Due to decades of extensive misuse and overuse of antibiotics in modern medicine, antibiotics that were once effective against pathogenic bacteria are now no longer sufficient because of rapid evolution and mutation of resistant bacterial species. Although novel antibiotics targeting MDR bacteria can be developed, pathogens ultimately end up becoming resistant to such drugs (Carlton 1999). To break this vicious cycle, phage therapy, which has been used extensively in former Soviet Union countries with success, is being reconsidered as a treatment method by Western researchers, who have exclusively relied on antibiotics to date. However, acceptance of this method has been difficult to obtain, and phage therapy must be subjected to rigorous clinical trials before it can be approved by the FDA as a treatment method (Gill et al. 2010; Abhilash et al. 2009). Phage Therapy History Phages were first discovered by British microbiologist Felix Twort in 1915, and later by FrenchCanadian microbiologist Felix d’Hérelle in 1917. Although Twort did not pursue his discovery, d’Hérelle investigated the nature and mechanism of phages as a therapeutic agent, and established phage therapy centers in the U.S., France, and Soviet Georgia (Carlton 1999). D’Hérelle’s first use of phage screening was on French troops with severe hemorrhagic dysentery in July 1915, where he made bacterium-free filtrates of patients fecal samples and incubated them with isolated Shigella strains from the patients. He observed the appearance of clear plaques on agar plates, which he proposed were caused by a virus capable of parasitizing bacteria. These phages were later used in phage therapy trials in 1919 on hospital patients with severe dysentery, and after a single dose, the patients fully recovered (Sulakvelidze et al. 2001). The use of bacteriophages as therapeutic agents was later used extensively during World War Two, particularly by Soviet doctors to treat wound infections of troops on the battlefield. However the discovery of penicillin in 1928 caused a sharp decline in phage research in the West, which chose to prioritize treatment 1 of bacterial infections with antibiotics, leaving only the former Soviet Union countries still developing and utilizing phage therapy (Abedon et al. 2011). Classification of Phages Phages are classed into 13 different families according to their morphology, presence or absence of an envelope or lipid, and type of nucleic acid. Approximately 96% of phages are composed of an icosahedral head and tail and have double stranded DNA as their genome and are termed “tailed phages.” Tailed phages are further classified by their morphological features into three families: Myoviridae, which possess a contractile tail and contain 93 species, including phages KVP20, KVP40, KVP241, and T-even; Siphoviridae, which possess a long non-contractile tail and contain 313 species, including phages ΦMR11 and λ; and Podoviridae, which possess an extremely short tail and contain 50 species, including T7 phages. The other 4% of phages are classified as cubic, filamentous, or pleomorphic and contain double stranded or single stranded DNA or RNA (Matsuzaki et al. 2005). Bacteriophage Life Cycle Phages are viruses that infect and lyse specific bacteria through interacting with bacterial membrane receptors, disrupting bacterial metabolism, and eventually causing the cell to lyse after replicating their DNA inside of the bacterial host cell. (Gill et al. 2010; Abhilash et al. 2009). Phages can be divided into two groups according to their life cycle: lytic phages, which insert their DNA into bacteria and self-proliferate, leading to bacterial lysis, and lysogenic phages, which have an additional lysogenic cycle where their DNA is incorporated into the host genome and replicated as part of the host genome without lysing the cell. However, under environmental stress, such as changes in temperature, pH, and nutrients, the lytic cycle can be triggered, lysing the cell. As some lysogenic phages have toxic genes in their genome which can become incorporated into the bacterial genome, lytic phages are the most suitable therapeutic candidates. Alternatively, pseudolysogeny can occur, in which the phage genome enters the cell but cannot enter the lytic or lysogenic cycle. This typically occurs in nutrient-deprived conditions, when bacterial hosts cannot support DNA replication, and the phage genome will persist as a preprophage until nutrition is restored, at which point it can enter the lytic or lysogenic cycle (Figure 1; Feiner et al. 2015:used with permission). 2 Figure 1. The lytic, lysogenic, and pseudolysogenic cycles. a. The lytic bacteriophage cycle, in which the phage replicates using host machinery and lyses the cell; b. The lysogenic cycle, where the phage genome is incorporated into the host chromosome as a prophage and persists in a dormant state until environmental stressors trigger the lytic cycle to commence; c. Pseudolysogeny, in which phage genome fails to replicate or establish itself as a prophage, occurring typically in nutrient-deprived conditions (Feiner et al. 2015, used with permission). 3 Mechanism of Bacteriolysis by Phages The first step of bacteriolysis is phage infection, through which phages adsorb to a receptor on the bacterial surface, which is typically a protein or sugar. Phages generally only adsorb to specific bacterial strains or species, not across multiple species or genera, which makes bacteriophage therapy as a targeted therapeutic treatment so beneficial. After phage adsorption, phage DNA is injected into host cytoplasm and is either integrated into the host chromosome or replicated by host machinery and packaged into capsids, which are created during the late stage of phage infection. Tails are then attached to the DNA-filled head. The new phages then lyse the cell through the protein interactions of lysin, which degrades peptidoglycan, and holin, which form holes in the cell membrane, exposing the peptidoglycan layers to lysin. The released phages infect other bacterial cells following this, leading to lysis of the entire bacterial population. (Figure 3; (Matsuzaki et ali. 2005: used with permission) Figure 2. Phage induced bacteriolysis: (1) Phage adsorption and DNA injection; (2) Phage DNA replication; (3) head and tail production; (4) holin and lysin synthesis; (5) DNA packaging; (6) complete phage assembly; (7) lysis of cell wall and release of phages; (8) circularization of phage DNA; (9) integration of phage DNA into host genome via lytic cycle (Matsuzaki et al. 2005, used with permission). 4 Bacteriophage Screening and Therapy Research Pseudomonas aeruginosa Vinodkumar et al. targeted 28 multidrug-resistant Pseudomonas aeruginosa strains in 1,647 septicemic mice using bacteriophages over a period of five years. Phages were isolated from raw sewage at a municipal sewage treatment plant after incubating sewage, nutrient broth, and P. aeruginosa at 58°C for 30 minutes in a water bath, following which a few drops of chloroform were added, the mixture was centrifuged, and the supernatant was filtered through a 0.22 µl filter. The effectiveness of bacteriophage activity on P. aeruginosa was confirmed in vitro through placing phage isolate in wells on a P. aeruginosa lawn for 24 hours at 37°C, using sterile distilled water as a control. The phage strain used was effective against 74% of the P. aeruginosa strains. The 28 multidrug-resistant strains were resistant to almost all types of antibiotics, including β lactamases, and were used to introduce a fatal infection into the mice. A single injection of the phage strain administrated 45 minutes after the P. aeruginosa injection was sufficient to rescue 100% of the animals, and an injection when the mice were moribund was successful in rescuing approximately 50% of the mice (Vinodkumar et al. 2008). A clinical trial by Wright et al. 2009 also tested phages against multiple-drug resistant P. aeruginosa in 24 patients with chronic otitis. Researchers found that the treatment patients showed significantly lower pathogenic P. aeruginosa levels 42 days after treatment compared to the placebo group and that no treatment-related adverse effect was reported. These results are indicative of phage therapy success in human clinical trials (Wright et al. 2009). Enterococcus faecium Biswas et al. 2002 used phage therapy on Vancomycin-resistant Enterococcus faecium infections in mice. Colonization of the gastrointestinal tract by Vancomycin-resistant E. faecium has become endemic in many hospitals and can lead to endocarditis, so a reliable treatment method is essential. Phages were isolated from municipal sewage through centrifugation and removal of the supernatant, following which the supernatant was added to precipitate in 10% polyethylene glycol, dissolved in SM buffer, and extracted with chloroform. This processed sewage was mixed with E. faecium and incubated for 20 minutes at 37°C, before being mixed with top agar and poured on 5 agar plates and incubated overnight. Phages present in plaques were isolated and used in mice given a potentially fatal E. faecium injection. Phage injection 45 minutes after pathogen injection resulted in the rescue of 100% of the animals, and phage injection into moribund animals resulted in approximately 50% of the mice being rescued (Biswas et al. 2002). Escherichia coli Chibani-Chennoufi et al. collected diarrhea-associated Escherichia coli samples from pediatric diarrhea patients and environmental water samples, and isolated phages from fecal samples through centrifugation and filtration of the supernatant through a 0.45 µl filter. Phage plaque assays were successfully performed, and purified phages were given to mice with regular and ampicillinresistant E. coli infections through drinking water. The results showed that some of the intestinal E. coli strains were lysed successfully, however, other strains were not. In particular, E. coli present in gut flora were only minimally affected by oral phage application, indicating that the phages were specific enough to only work against specific E. coli strains (Chibani-Chennoufi et al. 2004). Benefits of Bacteriophage Therapy As phages are composed mostly of nucleic acids and proteins, they are much less toxic than antibiotics, and phage therapy is seen as a superior method to antibiotics as it is much more specific and should be less likely to cause side effects or negative harm to the beneficial normal flora of the host (Abedon et al. 2011; Loc-Carillo et al. 2011; Matsuzaki et al. 2005). In addition, phages are reportedly very successful against bacteria that construct biofilms composed of a polysaccharide matrix that antibiotics cannot penetrate (Abhilash et al. 2009). Bacteriophages are also notably more successful at completely killing the target bacteria species in comparison to antibiotics, and are also capable of increasing in number in an area specifically where the bacteria species is located, therefore phages themselves contribute to establishing the phage dose during phage therapy. Because of this, only a single dose of phages is generally required for treatment (Loc-Carillo et al. 2011). Phages are also successful against extremely resistant bacteria strains. In addition to resistance to regular antibiotics, bacterial strains have been found to contain enzymes such as carbapenemases 6 and Extended-Spectrum Beta-Lactamases (ESBL), which further add to the broad resistance of the strains to treatment. Carbapenemases are β-lactamases which are capable of hydrolyzing carbapenems, cephalosporins, monobactams, and penicillins (Queenan et al. 2007). As multiple drug resistance (MDR) to antibiotics is easily passed through a plasmid during bacterial conjugation, carbapenems have been used to counteract MDR resistance, however this has led bacterial strains to develop resistance to carbapenems as well (Currie 2012). ESBLs are also βlactamases found only amongst Gram negative bacteria, and mediate resistance to extendedspectrum cephalosporins and monobactams, but do not affect cephamycins or carbapenems (CDC 2010). Infections from bacteria which possess these enzymes renders antibiotic treatment highly unsuccessful, illustrating the importance of the development of an alternative treatment method, such as phage therapy. In addition, phages are easily applied in the form of topical treatments and injections, and are versatile with regards to formulation development, as they can be used in combination with antibiotics or with other phage strains to increase the success of infection treatment (Loc-Carillo et al. 2011). Drawbacks of Bacteriophage Therapy The ideal phage is obligately lytic, stable in storage conditions and temperatures, subject to appropriate safety studies, specific to the target bacteria, and ideally, fully sequenced to ensure that it carries no toxin genes, which are difficult conditions to meet (Loc-Carillo et al. 2011). Even if a specific phage that is effective is found, it must be able to enter the lytic cycle to be used in treatment, as lysogenic phages will lay dormant until environmental stress is experienced by the host cell. Phages must also be able to be stored for long periods of time after they are isolated without breaking down, typically at -20°C (Prescott et al. 2005). Another issue is getting approval to put phage therapy on clinical trials so that it can be accepted as a treatment method in the west. Former Soviet countries do not face this problem, but for phage therapy to be put into use and approved by the FDA, rigorous clinical trials must be undergone. In addition, changing the western mindset to accept using viruses to treat infections presents another challenge. Phages only attacking a narrow host range is another problem, as isolating phages specific-enough for certain bacteria may take a lot of time and energy. Because of this, multiple phages may need to be 7 combined in a dosage in a so-called “phage cocktail” in order to most effectively target the bacteria (Loc-Carillo et al. 2011). Finally, phages have been known to contain toxin genes and regulate virulence in bacteria, so sequencing phages before they are put to therapeutic use is essential to ensuring the treatment will be beneficial (Wagner et al. 2002). Bacteriophage Therapy in Combination with Other Treatment Methods Although phages are used exclusively as a treatment method in Russia, countries like Poland use phages in combination with other treatment methods, in order to maximize treatment potential (Carlton et al. 1999). Although resistance to common antibiotics has become an issue, researchers are still finding novel antibiotics to which bacteria do not have resistance genes, which can potentially be used in combination with phages to ensure that all pathogenic bacteria are killed (Huff et al. 2004). Another novel treatment method exclusive to antibiotics and phage therapy is using clay to kill bacteria, as exhibited by the Kisameet Clay in the Heiltsuk First Nation territory in British Columbia, which has been shown to have antibacterial activity against MDR Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumonia, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species (Behroozian et al. 2016). Phages could potentially be used in combination with this clay to give the most effective treatment possible for bacterial infections, much like the combination of antibiotics and phages. SOURCES OF BACTERIOPHAGES Water Samples Used Using water samples from the Kamloops Sewage Treatment Plant, Domtar, the Pacific Ocean, Bisaro Anima Cave, alkaline ponds, and the non-water dirt sample in this project as a source of phages could have allowed for the discovery of novel phages which could have been extremely successful in lab. For instance, sewage contains many potentially pathogenic bacteria, such as Escherichia coli and Enterobacter aerogenes, which suggests that phages capable of successfully attacking these potentially dangerous and rapidly-evolving bacteria may also be present in the sewage sample from the Kamloops Sewage Treatment Center (Beaudoin et al. 2007). Likewise, cave water and Domtar runoff present extreme habitats, seeing as bacteria are forced to grow in energetically unfavourable and nutrient-limited conditions, and may host many novel and diverse 8 bacteria species and their respective phages, which may be effective against other MDR bacteria (Barton et al. 2007). In addition, alkali pond water is extremely acidic and selects for resistant and adaptive bacteria such as E. coli. We can speculate that if E. coli is present excessively in alkali pond water, phages associated for E. coli are likely present as well (Parhad et al. 1974). Seawater has also been found to contain over 150 different isolates of bacteriophages and phages are shown to exceed bacterial concentration in seawater by a factor of 10, therefore seawater should have been a rich source of phages (Børsheim 1993). Soil was also speculated to contain bacteriophages effective against MDR bacteria, as soil contains many bacteria that can become pathogenic to humans, and an average of 1.5 x 108 g-1 phages, which is equivalent to 4% of the total population of bacteria (Ashelford et al. 2003). The creation of an enrichment water sample was also attempted through the incubation of broth culture, nutrient broth, and filtered sewage water overnight at 37°C. Through exposing the bacteriophages present in the water sample to only one bacterial strain, it was projected that more successful phage screening trials would occur (Biswas et al. 2004; Chibani-Chennoufi et al. 2004; Prescott et al. 2005; Wright et al. 2009; Vinodkumar et al. 2008). The successfulness of each environmental water sample was measured through the formation of plaques, which develop after plating the MDR bacteria, molten agar, and environmental water for confluent growth (Prescott et al. 2005). The development of plaques on the E. coli strains would further signal that bacteria possessing carbapenemases and ESBLs and are essentially untreatable can be treated successfully using phages. All plaques were sent to sequence the bacteriophages present to determine if novel phages had been found. OBJECTIVE The main objective of this project was to screen bacteriophages (bacteria-infecting viruses) from extreme habitats that specifically target the multiple drug resistant (MDR) bacteria. In particular, this experiment focused on the isolation of phages from local water samples, namely sewage water samples from the Kamloops Sewage Treatment Plant, and their effectiveness against MDR E. coli obtained from LifeLabs. We aimed to develop insight into the correct process, optimal concentrations, and proper conditions needed for phage isolation, and determine whether 9 successful bacteria-specific phages were capable of being screened, grown, and used against MDR bacteria. The null hypothesis was that the MDR bacteria would be unaffected by bacteriophage screening and live regardless of the inoculation with phages. MATERIALS AND METHODS WATER SAMPLE COLLECTION Water samples were obtained aseptically in sterile bottles from the Kamloops Sewage Treatment Plant, Domtar (three samples taken at 1.4 days, 2.3 days, and 3.3 days into the purification process, with the sample taken at 3.3 days being the final treated effluent), the Pacific Ocean (Bamfield, B.C.), Bisaro Anima Cave, and alkaline ponds. A water sample was also created from mixing dirt from Abbotsford, B.C. with sterile water. All samples were stored at 4°C until use. WATER SAMPLE FILTRATION FOR BACTERIOPHAGE ISOLATION Water samples were either syringe-filtered or pressure-filtered through a 0.22 µm filter to remove bacterial particles, ensuring only phages were present. Samples containing a lot of particulate and debris were first filtered with a 0.45 µm filter, and then a 0.22 µm filter. In one trial, an enrichment water sample was created through the combination of nutrient broth, E. coli 14-318 bacterial culture, and sewage water incubated overnight at 37°C to promote the growth of strain-specific phages, before being filtered through a 0.22 µm filter. The soil sample from Abbotsford was mixed with sterile water and let stand for 24 hours. It was then filtered using a 0.22 µm filter. CONTROLS Positive and negative controls were used in the experiment. For the negative control, sterile water was plated in combination with the MDR bacteria, expecting that the bacteria would exhibit confluent growth and no plaque development would occur. The positive control involved plating the environmental water samples with a non-MDR strain of E. coli that was previously shown to be killed by phages present in the Kamloops Sewage Treatment Centre water sample, expecting that plaques would develop in this situation. 10 SEQUENCING A plate containing plaques from sewage water which was successful against E. coli 14-318 was sent to GENEWIZ, Inc, in Seattle, USA, for sequencing. Following unsuccessful sequencing, new plates were sent to the University of Texas for sequencing. Both times, the standard laboratory procedure was repeated to ensure plaque formation, following which the fresh plate was wrapped in parafilm and shipped via express post on ice. SAFETY PRECAUTIONS The experiments were undergone in the laboratory in a biosafety cabinet so as to not contaminate other areas of the lab with the water samples and the MDR bacteria. All contaminated equipment was labelled and kept together and all samples were labelled and stored in an isolated area in the walk-in fridge. Proper biosafety training was also undergone in accordance with the requirements for Laboratory safety 2. 11 PROCEDURE Figure 3. Overview of experimental procedure outlining the isolation and purification of bacteriophages from water samples, growth of bacteria in nutrient broth, and combination of phage water, bacteria, and molten agar on nutrient agar plate. MDR Bacteria Preparation Bacteria strains were confirmed to be multiple-drug resistant through a streak plate test on nutrient agar plates infused with tetracycline. Once resistance was confirmed, bacterial species were inoculated in nutrient broth overnight, shaking at 37°C, and then placed in the fridge to preserve cell density. Isolation and purification of phage strains Water samples were stored in the fridge at 4°C until ready for use. Samples were first filtered through a 0.45 µm filter to remove bacteria and excess debris if needed, followed by a 0.22 µm 12 filter, to leave only bacteriophages present in the water samples. The environmental water was stored at 4°C in fridge until needed. In vitro confirmation of bacteriophage activity In a test tube, 0.5 mL environmental water and 0.5 mL broth culture bacteria were mixed with 2.5 mL molten nutrient agar warmed to approximately 55°C, which was hot enough to pour easily but not hot enough to kill the bacteria, and then poured evenly onto nutrient agar plate. This step was also repeated with dilutions of 0.9 mL environmental water and 0.1 mL bacteria, but no significant differences in plaque formation were seen between the two dilutions. Petri plate lids were immediately placed onto plates to avoid contamination, and the plates were inverted and incubated at 37°C for 24 hours. After incubation, the plates were checked for the presence of plaques. If plaques were found, the procedure was repeated again to ensure legitimate plaque formation, and the plates were wrapped in parafilm and sent via express post to be sequenced. RESULTS The combination of top molten agar, broth culture, and filtered environmental water samples was first attempted using the water samples from the Kamloops Sewage Treatment Center, the Pacific Ocean, and Domtar, however no plaques formed. The same protocol was repeated using strainspecific environmental water samples through the incubation of broth culture, nutrient broth, and environmental water overnight before filtering for isolated phages, however no plaque development was observed. Furthermore, plaque formation was not seen using water samples from Bisaro Anima Cave, alkali ponds, or the soil sample (Table1). A more crude water sample was obtained from the Kamloops Sewage Treatment Centre, from which phages capable of successfully killing the MDR bacteria were only found. The only bacterial strain that developed plaques from this water sample was the E. coli 14-318 strain at both a 5:5 dilution and a 9:1 dilution of sewage water to broth culture (Table 2). A sewage water enrichment sample was prepared through the inoculation of broth culture in sewage water and nutrient broth overnight to induce strain-specific phages and was successful against the E. coli 14-318 strain. For the 5:5 broth dilution, 31 plaques were found, ranging in sizes from 1 mm to 5 mm and for the 5:5 13 sewage dilution 22 plaques were observed, sized 2 mm to 7mm. For the 9:1 broth dilution, 52 plaques were found ranging in size from 0.5 mm to 11 mm, and for the 9:1 sewage dilution, 39 plaques were noted from 1mm to 7 mm (Table 3). Overall, no significant difference was noted in the successfulness of phages between those present in the enrichment water and those in the sewage water, indicating that the enrichment water did not contain more strain-specific phages, and that the phages were equally as successful at lysing bacterial cells. 14 Table 1. The resistance, possession of carbapenemase and ESBL, and plaque presence observed of the MDR bacteria used in this project. Strain Resistance Carbapenamase ESBL Plaque Presence E. coli 15-102 Ampicillin1 + + - E. coli 15-124 Ampicillin2 + + - E. coli 14-318 Ampicillin3 + + + Micrococcus luteus TET (tetracycline-30 µg) U5 U - MRSA TET (tetracycline-30 µg) U U - U U - MRSA 1 Serratia Tetracycline4 U U - Mybacterium SXT (sulfamethoxozole-23.75 U U - smegmatis µg and trimethoprim-1.25 µg) Control : E. coli None U U + Ampicillin, Amoxicillin/Clavulanic Acid, Piperacillin/Tazobactam, Cefalotin, Cefazolin, Cefoxitin, Cefixime, Ceftazidime, Ceftriaxone, Ertapenem, Meropenem, Amikacin, Gentamicin, Tobramycin, Ciprofloxacin, Tetracyclin, Nitrofurantoin, Trimethoprim/Sulfamethoxazole, 2 Ampicillin, Amoxicillin/Clavulanic Acid, Piperacillin/Tazobactam, Cefalotin, Cefazolin, Cefoxitin, Cefixime, Ceftazidime, Ceftriaxone, Ertapenem, Meropenem, Ciprofloxacin, Tetracyclin, Trimethoprim/Sulfamethoxazole, 3 Ampicillin, Amoxicillin/Clavulanic Acid, Piperacillin/Tazobactam, Cefalotin, Cefazolin, Cefoxitin, Cefixime, Ceftazidime, Ceftriaxone, Ertapenem, Meropenem, Amikacin, Gentamicin, Tobramycin, Ciprofloxacin, Tetracyclin, Nitrofurantoin, Trimethoprim/Sulfamethoxazole, 4 Tetracycline, Gentamicin, Nitrofurantoin, Ceftriaxone, Tobramycin, Amikacin, Cefixime, Meropenem, Ertapenem 5 Unknown 15 Table 2. The water sample location and presence of plaques when water samples had been plated with MDR bacteria and molten agar. 1 Water Sample Location Plaque Presence Alkali Ponds No Bisaro Anima Cave No Domtar (three different samples) No Kamloops Sewage Treatment Plant Yes1 Sea Water (Bamfield, B.C.) No Soil Sample (Abbotsford, B.C.) No Worked against E. coli 14-318 and control E. coli Table 3. The different water sample (enriched or regular sewage) to bacterial broth dilutions and the number and size of plaques present. Water Sample Dilution Number of Plaques Plaque Size 500 µL Enrichment : 500 µL Bacteria 31 1 mm - 5 mm 500 µL Regular : 500 µL Bacteria 22 2 mm - 7 mm 900 µL Enrichment : 100 µL Bacteria 52 0.5 mm - 11 mm 900 µL Regular : 100 µL Bacteria 39 1 mm - 7 mm 16 Table 4. Additional resistance of E. coli 14-318 to beta lactams, aminoglycosides, quinolones, tetracyclines, furanes, and trimethoprim/sulfonamides. Antibiotic Types Resistance Beta Lactams ESBL, including carbapenemase Aminoglycosides Gentamicin, Tobramycin, Netilmicin Quinolones All Tetracyclines All Furanes All Trimethoprim/Sulfonamides All 17 Figure 4. Plaque presence after plating bacterial strain E. coli 14-318 with molten agar and sewage water and sewage broth samples in different concentrations: 1. 500 µL sewage water to 500 µL bacterial culture; 2. 500 µL enrichment sewage to 500 µL bacterial culture; 3. 900 µL sewage water to 100 µL bacterial culture; and 4. 900 µL enrichment sewage to 100 µL bacterial culture. 18 DISCUSSION The absence of plaques found from plating a combination of top agar, broth culture, and filtered environmental water samples prompted the need for the growth and isolation of more strainspecific phages. This led to the creation of an enrichment broth through the inoculation of water samples, broth culture, and nutrient broth overnight before filtering the water for isolated phages. The continued absence of phages inferred that either phages were not present in the water samples, or that phages specific enough to kill the MDR and control bacteria were not present. The latter point is supported, as the phages present in sewage water were successful against both the E. coli 14-318 strain and the control E. coli strain, however not against the E. coli 15-102 and 15-124 strains, indicating the specificity required for phages to attack a cell, even amongst bacteria of the same species (Chibani-Chennoufi et al. 2004). In addition, as sewage contains human excrement, which is rich in E. coli and other human pathogens, it was expected that phages capable of killing human pathogens would be present in the samples. However, the other water samples used came from environments that are not necessarily rich in human pathogens, which could account for the lack of phage specificity and plaque development. A more crude sewage water samples was then put through the protocol, both as a standard filtered water sample and also as an enriched water sample, which was prepared through the incubation of broth culture, nutrient broth, and sewage water overnight. Sewage from sewage treatment centers is a rich source of phages and is used extensively in protocols in previous research as it contains a wide variety of human gut bacteria, potentially containing phages which specifically target human pathogens like the MDR bacteria used in the study. Due to the wide variety of bacteria present in sewage, many different bacteriophages could be present in one sample, which increases the chances of more MDR bacteria being targeted by bacteriophages. (Biswas et al. 2002; Vinodkumar et al. 2008; Wright et al. 2009). Phages have also been isolated from patient feces in the previous research, however only using feces from one subject likely narrows the amount of phages isolated (Chibani-Chennoufi et al. 2004). 19 Plaque presence was observed in both the E. coli 14-318 strain and the control E. coli strain, and there was not a noticeable difference in the number of plaques between the regular sewage sample and the enriched sample, indicating that the enriched sample did not improve specificity. These results were not expected, as enriched environmental water samples are used in most studies for phage screening, and were proposed to be more specific and successful against pathogens. Plaque size, however, differed considerably on the plates, ranging from 0.5 mm to 11 mm, potentially indicating that different phages were present (Biswas et al. 2004; Chibani-Chennoufi et al. 2004; Prescott et al. 2005; Wright et al. 2009; Vinodkumar et al. 2008). The last water samples tested were from Bisaro Anima Cave and alkaline ponds, however no plaques developed after plating. Soil was also used as a phage source after being mixed with sterile water and filtered, but no plaques developed after plating. Along with the first water samples tested, it was inferred that phages did not exist in the water samples or that phage screening was not successful against the specific MDR bacteria. Compared to sewage water samples, which contain human excrement containing gut microbiota and potentially pathogens, extreme water and soil samples such as the Bisaro Amina Cave, alkali ponds, and soil samples used in this project are not necessarily exposed to the same species and strains of bacteria that associate with humans. Although the presence of E. coli in alkali ponds and soil samples has specifically been supported in previous studies, perhaps the phages present in these samples were not specific to the MDR E. coli used in this project (Børsheim 1993; Parhad et al. 1974). This postulation is supported as phages effective against one MDR E. coli strain were not effective against all of the MDR E. coli strains used, further illustrating the importance of specificity in phage screening. Indeed, the need for species- and strain-specific phages hinders the development of phage therapy as a therapeutic treatment method as such phages can be difficult to isolate and discover, rendering some bacterial infections untreatable. Although antibiotics negatively affect good gut microbiota, they are designed to kill all bacteria without needing specific parameters or specificity to do so, making antibiotics a broader and more effective treatment method theoretically. However, in the case of MDR bacteria, where antibiotics are useless, the time and energy spent finding phages specific to these bacteria is extremely important and critical to the treatment of these bacteria (Loc-Carillo et al. 2011). 20 Plates with E. coli 14-318 plaques were sent to be sequenced at GENEWIZ, Inc. Although a phage primer was used, phage genomes do not have the same conserved regions that bacterial genomes have, therefore the primer did not bind well to the E. coli 14-318 genome and the sequencing results were inconclusive (Hattful 2008). The plaques are now being sequenced at the University of Texas. The results of this experiment were significant, as phages capable of killing MDR E. coli through phage screening were found to be present in local sewage and successful in multiple trials. Furthermore, the E. coli 14-318 strain possesses beta lactamases and is resistant to aminoglycosides, quinolones, tetracyclines, furanes, trimethoprims, and sulfonamides, which make antibiotic treatment nearly impossible. Therefore, the discovery of an alternative method to kill this bacteria strain is significant as no other common treatment method has been successful. In addition, these results contribute positively to phage therapy research and clinical trials in North America, and continued results of these trials will hopefully result in the FDA approving phage therapy as an alternative treatment method. For future expansion of this research, more environmental water samples should be collected, including sewage and fecal samples from other cities and countries, considering feces is rich in phages specific to human pathogens and diverse sample sites could provide a wider range of phages. Phages could also be isolated from compost, landfills, and soil samples which are exposed to human wastes. In addition, more E. coli strains should be collected and screened to observe which strains the sewage phages are effective against, in order to further investigate the specificity of bacteriophages amongst a species. Although there are downsides to phage therapy, such as the requirement for phages to be obligately lytic, stable in storage, safe in experimental studies, matched to specific bacteria, and fully sequenced to ensure they won’t pass on toxicity, phage therapy is an effective therapeutic treatment method to treating bacterial infections, and could be used against bacteria which are resistant to antibiotics. This method has been used extensively in the former Soviet Union countries with success and is currently being researched extensively in the United States before it can be placed in clinical trials to be approved by the FDA as a treatment method. Along with phages being more 21 specific to target bacterial species and not having a negative effect on beneficial host microbiota, only one dose is needed for treatment as phages regulate their numbers based on the amount of target bacteria present and phages can be combined easily with other treatment methods, such as antibiotics and clay, to ensure the most effective and efficient treatment of bacterial infections. Although time, money, and energy need to be put into phage research and clinical trials, approval of this treatment method may be crucial for western medicine to counter the epidemic of antibiotic resistance. The benefits of phage therapy clearly outweigh the disadvantages. 22 LITERATURE CITED Abedon ST, Kuhl SJ, Blasdel BG, Kutter EM. Phage treatment of human infections. Bacteriophage [Internet]. 2011 [cited 2016 Apr 2];1(2):66–85. Available from:http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3278644/ Abedon S, Loc-Carrillo C. 2011. Pros and cons of phage therapy. 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