University of Nevada, Reno
Isolation of bacteriophages against Streptococcus species; Lysogeny of Ten Mycobacteriophages for host Mycobacterium tuberculosis H37Ra.
A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Environmental Sciences
by
Christine B. Emmons
Dr. Laura Briggs/Thesis Advisor
May 2020
THE GRADUATE SCHOOL
We recommend that the thesis prepared under our supervision by CHRISTINE B. EMMONS
ntitled Isolation of bacteriophages against Streptococcus species; Lysogeny of Ten Mycobacteriophages for host Mycobacterium tuberculosis H37Ra be accepted in partial fulfillment of the requirements for the degree of
Master of Science
Laura Briggs, Ph.D. Amilton de Mello, Ph.D. Co Jonathan Baker, Ph.D.
May, 2020
i
Abstract
Bacteriophages are viruses that infect bacteria, often resulting in lysis of the bacterial host. Due to the specific nature of bacteriophages, they play an essential role in maintaining the microbial balance of ecosystems. Truckee
Meadows Community College has been isolating bacteriophage from soil and water samples in conjunction with a national undergraduate research program since 2014. The focus of Truckee Meadows Community College research has primarily been on mycobacteriophages; specifically, those that infect
Mycobacterium smegmatis mc2155. Two projects are presented in this thesis.
The first is the methods to capture and isolate Streptococcus bacteriophages using human saliva tested against Streptococcus mutans lab strains, and other wild-type bacteria obtained from the human oral cavity. The second is lysogeny of mycobacteriophages originally obtained from Mycobacterium smegmatis mc2155 as a host that displayed successful cross-infection with Mycobacteria tuberculosis H37Ra in previous assays. Using the previous research, the bacteriophages capable of cross-infectivity were tested for their ability to form a lysogen – to integrate viral DNA within the genome without killing or lysing the bacterium; thus, resulting in bacteriophage readiness to take over the host cell in the current bacterium, as well as future generations of the bacterium.
Bacteriophages demonstrate high specificity to their host. Receptors for bacteriophage to bind to the host bacterium appear to be in the cell wall of Gram- negative and Gram-positive bacteria, through the capsule and slime layer, and ii through appendages like flagella. Though phage-specific receptors have not been identified. The tail fibers of the bacteriophage appear to be significant in finding those receptors, but the specific genes responsible also have not been identified. Developing a process for successful isolation of bacteriophage is crucial to the process of understanding specificity through characterization of the bacterial host and the bacteriophage isolated.
In the Streptococcus bacteriophage study, eight lab strains of
Streptococcus mutans and 11 wild-type bacteria were cultured from the human oral cavity, cultured were used as bacterial hosts. The Neisseria strains were removed from this research. Saliva was tested against the host to test for the presence of novel bacteriophage. Initial methods were derived from previously published methods for Streptococcus mutans bacteriophage isolation.
Bacteriophage isolation was not successful using these initial methods, so several modifications were tested. Modifications included an increase of infection time, incubation time, limiting potential bacteriophage tail damage from centrifugation – reduced centrifugation times or elimination of centrifugation, using saliva without filtration, and using saliva without filtration and not allowing the sample to settle. While no bacteriophage was successfully isolated for characterization, these procedural changes did result in potential temperate phages; this indicates a potential for temperate bacteriophage isolation in future studies.
In the mycobacteriophage study, previous research identified ten mycobacteriophages with the potential for cross-infectivity between the original iii host, Mycobacterium smegmatis mc2155, and the potential host, Mycobacterium tuberculosis H37Ra. This research confirmed that six mycobacteriophages were successful in cross-infection: Scooby Blue, Erimy, Guilian 2, Guilsminger, Old
House, and Zose. Five were lysogenic, and one was lytic. The lytic bacteriophage, Zose, was not used for this study. Two of the remaining five mycobacteriophages showed an efficiency of plating (EOP) of 10-3 or better:
Scooby Blue and Old House when compared to the original host. An EOP of 10-3 or better indicated productive infection of these bacteriophages, which makes these two bacteriophage potential candidates for characterization of the genomes for further understanding of cross-infectivity and lysogeny.
Future work should include further adjustments to protocols for more successful isolation of Streptococcus bacteriophages. The adjustments should include longer enrichment times and sampling from other environmental sources, including soil. Future studies using the lysogenic mycobacteriophages analyzed here should include DNA sequencing of the mycobacteriophages for comparison of each genome against lytic and lysogenic bacteriophages that cross-infect original host, Mycobacterium smegmatis mc2155, and the potential host,
Mycobacterium tuberculosis H37Ra, allowing for identification of lysogeny genes.
The same bacteriophage genomes can be used for comparison with other mycobacteriophage genomes that do not cross-infect to specify genomic differences specific to cross-infection.
iv
Acknowledgments
This research was made possible because of many individual contributions, some to the research and others to me personally. First, I would like to thank my
Advisor, Dr. Laura Briggs. Her dedication to research, passion for teaching, and guidance through this process has been invaluable to me as a student, teacher, and researcher. I have been privileged with many opportunities throughout my career as a student, and it is in large part because she believed in me and my abilities, thank you, Dr. Briggs. I would like to thank Dr. Meeghan Gray. Her passion for teaching started me on a scientific path. Her valuable insight as a
Professor, peer, and friend has helped me get to where I am today, thank you,
Dr. Gray. I would like to thank Dr. Julie Ellsworth and Tina Slowan-Pomeroy for allowing me to continue to teach, research, go to school, and keep working in the lab. It has provided me the opportunity to get where I am today, thank you both.
Thank you to my committee members, Dr. Amilton de Mello and Dr. Josh Baker, for guiding me through this process and always offering to help in any way that you can.
My research was possible because of Nevada INBRE and Truckee
Meadows Community College. Nevada INBRE provided the grant that got this research started, and Truckee Meadows Community College allowed me to do graduate research at the facility. v
Finally, I would like to thank my husband, Thomas Emmons, and my son,
Tristian Sommerfield. The constant support and love helped me through my education, and I am forever grateful for your belief in me, thank you.
vi
Table of Contents Abstract ...... i Acknowledgments ...... iv List of Abbreviations ...... viii List of Tables ...... x List of Figures ...... xi Chapter 1 ...... 1 Significance ...... 2 Biofilms ...... 3 Streptococcus mutans ...... 4 Infective Endocarditis ...... 5 Mycobacterium tuberculosis ...... 5 Tuberculosis History ...... 7 Bacteriophage History ...... 8 Bacteriophage Diversity ...... 9 Bacteriophage Anatomy and Lifecycles ...... 10 Bacteriophage Specificity and Host Range ...... 12 Bacteriophage Treatment Today ...... 13 Lytic and Lysogenic Bacteriophages for Treatment of Infection ...... 14 Summary ...... 16 References ...... 18 Chapter 1 Figures ...... 20 Chapter 2 ...... 22 Abstract ...... 25 Introduction ...... 25 Materials and Methods ...... 27 Results ...... 36 Discussion ...... 36 Acknowledgments ...... 39 Authorship Confirmation Statement ...... 40 References ...... 42 vii
Chapter 2 Tables ...... 43 Chapter 3 ...... 47 Abstract ...... 50 Introduction ...... 50 Materials and methods ...... 55 Results ...... 59 Discussion ...... 60 Future Directions ...... 62 Acknowledgments ...... 63 Authorship Confirmation Statement ...... 64 References ...... 66 Chapter 3 Figures ...... 68 Chapter 3 Tables ...... 70 Chapter 4 ...... 72 Conclusions ...... 73 Future Directions ...... 76
viii
List of Abbreviations
AD: Albumin Dextrose
ADC: Albumin Dextrose Catalase
CaCl2: Calcium chloride
DAOSA: Double agar overlay spotting assay
DAOSA7H9: Double agar overlay spotting assay with 7H9 Media
DAOSABHI: Double agar overlay spotting assay with BHI
EOP: Efficiency of Plating
HD: Hardy Diagnostics
IE: Infective Endocarditis
IPATH: Center for Innovative Phage Applications and Therapeutics
LA: Luria Agar
LPS: Lipopolysaccharide layer (LPS)
M. smegmatis: Mycobacterium smegmatis mc2155
M. tuberculosis: Mycobacterium tuberculosis H37Ra mL: milliliters
NAD+: Nicotinamide adenine dinucleotide nm: nanometers
OACD: Oleic Albumin Dextrose Catalase
PCBSDBHI: Phage capture by serial dilution with BHI
Phage(s): bacteriophage(s)
R. mucilaginosa: Rothia mucilaginosa
S. mutans: Streptococcus mutans ix
SEA-PHAGE: Science Education Alliance-Phage Hunters Advancing Genomics and Evolutionary Science
TB: Tuberculosis
TMCC: Truckee Meadows Community College
TNT: Tuberculosis Necrotizing Toxin
µl: microliters
x
List of Tables
Table 1: NCBI BLAST results for all bacteria tested with this project...... 43 Table 2: Results of original protocol for phage capture via human saliva enrichment...... 43 Table 3: Method 2 Results (Termination of Centrifugation) for phage capture via human saliva enrichment...... 44 Table 4: Method 3 results (Addition of CaCl2 & Continuous Saliva Collection) for phage capture via human saliva enrichment...... 44 Table 5: Method 4 results (Addition of glycerol) for phage capture via human saliva enrichment...... 45 Table 6: Potential bacterial hosts tested during cross infectivity assay ...... 45 Table 7: Results of host range assay of phage that spotted in triplicate. Phage host listed on the top line...... 46 Table 8: Results for the Bacteriophage capture via soil enrichment...... 46 Table 1:9Efficiency of Plating (EOP) for all mycobacteriophages tested that can cross infect M. tuberculosis compared to the original host, M. smegmatis...... 70 Table 2:10Ten bacteriophage with cross infection capabilities tested for lysogeny. Scooby Blue and Old House bacteriophages spotted, produced mesas, and bacterial clearings on both M. tuberculosis and original host bacteria M. smegmatis making 2 of the 10 tested (20%) potential lysogens for M. tuberculosis and M. smegmatis...... 71
xi
List of Figures
Figure 1: Bacteriophage Order Caudovirales. Myoviridae: long contractile tails, Podoviridae: short non-contractile tails, and Siphovirdae: long non-contractile tails https://www.pngwave.com/png-clip-art-wluvj ...... 20 Figure 2: Bacteriophage Structure https://viralzone.expasy.org/resources/pro_VIRION_phage.jpg ...... 20 Figure 3: Upon attachment and injection of genetic material the bacteriophage enters a lytic or lysogenic lifecycle. https://www.researchgate.net/post/please_mention_the_the_main_stages_of_a_ phage_life_cycle_and_can_explain_the_major_difference_between_the_lytic_an d_lysogenic_cycle ...... 20 Figure 4: Presence of clear plaques on agar plates indicate a phage in a lytic lifecycle. http://www.sci.sdsu.edu/~smaloy/MicrobialGenetics/topics/phage/phage-lambda- plaque.gif ...... 20 Figure 5: Presence of cloudy or turbid plaques on an agar plate indicate a phage in the lysogenic lifecycle.https://www.researchgate.net/profile/David_Hopwood/publication/18579 974/figure/fig3/AS:601688721342478@1520465178735/Three-day-old-plaques- of-the-temperate-phage-VP5-on-S-coelicolor-A32-showing-a-clear.png ...... 21 Figure 6: Components of lipopolysaccharide and other endotoxins http://1.bp.blogspot.com/_FN1wR3ASuIU/TB32KBUdvbI/AAAAAAAAB9Q/uODU TIKS9v8/s400/2.jpg ...... 21 Figure 7: Gram-negative endotoxin release upon cell death. http://2.bp.blogspot.com/_FN1wR3ASuIU/SnXz_GvaoPI/AAAAAAAAAjg/i3q4J_d Bqq4/s400/bacterial+toxins.jpg ...... 21 Figure 1:8Comparison of phage titer on M. smegmatis and M. tuberculosis. .... 68 Figure 2:10Phage Efficiency of Plating (EOP) on M. smegmatis compared to M. tuberculosis. Of the 20 phages plaque tested for infection efficiency, 30% were capable of infecting M. tuberculosis within an efficiency threshold of 10-3: Peachy, Guilian2, OldHouse, Erimy, ScoobyBlue, and Warrosco; 20% were able to infect M. tuberculosis at a reduced rate: Guillsminger, Alishanda, Lazanducci, and Zose. Of the 20 phages tested for infection efficiency, 50% were not able to infect M. tuberculosis via the plaquing method: Battleborn, Chandler, ChandlerG, DeeCee, Idlewild, IdlewildPark, Manaxia, Mistake, and Sierra15...... 69
1
Chapter 1 Introduction
2
Significance
Antibiotics are compounds produced by both bacteria and fungi that inhibit the growth of certain bacterial species. The initial discovery in 1929 by
Alexander Fleming of "mold juice" was just the beginning of the widespread use and manufacturing of antibiotics in the treatment of infectious disease. In 1944
Pfizer founded the first commercial large-scale production plant of penicillin, and the era of antibiotics began (Discovery and Development of Penicillin, 2019).
Since 1944 treatment of infectious disease has been most effective. However, excessive use of antibiotics has started a new, more important than ever before, concern of antimicrobial resistance (Neu, 1992). New resistance mechanisms are arising and spreading every day, and previously treatable diseases like pneumonia, tuberculosis, blood poisoning, gonorrhea, and foodborne disease are, in some cases, not treatable due to antibiotic resistance. Antibiotic resistance is the product of the evolution of bacteria. These evolved changes in the bacteria prevent previously successful antibiotic treatments from being effective against the new, evolved bacteria. While newly synthesized methods are being tested, antibiotic resistance persists, and other treatment options for bacterial infection and disease need to be considered (World Health
Organization, 2018).
Antibiotic treatments target specific mechanisms like cell wall synthesis, protein synthesis, cell membrane function, and nucleic acid synthesis in bacteria.
Due to the generality of those mechanisms, they exist in many different bacteria, mutualistic, commensal, and parasitic; thus, resulting in bacterial death of 3 bacteria needed for biological function and those that cause infection and disease. A potential alternative to such a broad treatment option is a more specific one, bacteriophages. Bacteriophages are viruses that specifically target bacteria, not the animal host. A bacteriophage is highly specific to the bacterial host and can be used to target the bacteria responsible for the infection and disease, not our mutualistic and commensal microbiota.
Biofilms
Many bacteria share a common attribute, the ability to form a biofilm. A biofilm is an involved community of bacteria held together by an extracellular matrix. Biofilms can occur in almost any environment; they have been found in environmental, industrial, and medical settings, in the human body on teeth, skin, and the urinary tract. The formation of a biofilm protects the bacteria from host defenses and exhibits resistance to antimicrobials (Daniel Lopez, 2010). Biofilms are associated with many different infections, and antibiotic treatment becomes difficult due to tolerance mechanisms provided by the extracellular matrix. The biofilm protection is called phenotypic resistance. Phenotypic resistance can also lead to genetic resistance, the alteration of the genetic code, with antibiotic exposure. When antibiotics are used, resistant bacteria are not harmed and can share their genes with other, non-resistant bacteria. Protection from the biofilm allows this transfer to happen more effectively (Oana Ciofu, 2017). Phenotypic resistance, genetic resistance, and environmental pressures can result in bacteria within biofilms that cannot be treated with modern antibiotics. Though many antibiotics are unable to penetrate the biofilm created by these bacteria, 4 bacteriophages have been shown to reduce biofilm mass, thus penetrating the biofilm and effectively killing the bacteria within. Bacteriophage can carry or express depolymerizing enzymes that breakdown the extracellular polymeric substance created by biofilm-forming bacteria (David R. Harper, 2014). One study indicated Pseudomonas aeruginosa, a Gram-negative biofilm producer, to have a reduction in biofilm mass by a median of 76% in 48-hours with the application of bacteriophage (Stephanie A. Fong, 2017), this indicates a new, more effective treatment of biofilms.
Streptococcus mutans
Tooth decay, also known as dental caries, is a breakdown of the teeth from acid-producing pathogenic bacteria. A meaningful contributor to dental caries is Streptococcus mutans. S. mutans is a viridans group, Gram-positive, facultatively anaerobic, biofilm-forming, cocci bacteria commonly found in the biofilm of the oral cavity of humans. Many viridans group streptococci constitute normal flora of the respiratory and gastrointestinal tract of humans.
Streptococcus mutans, within that group, is a significant pathogen and considered a causative agent of infective endocarditis (IE). Streptococcus mutans can enter the bloodstream from oral trauma such as dental procedures, daily oral care, or other injuries. Once the bacterium is in the bloodstream, individuals with underlying heart conditions are at risk for IE; healthy individuals will clear the bacterium effectively with the immune system (Masatoshi Otsugu,
2017). One defense mechanism of S. mutans is the creation of a biofilm; this protects the bacteria from external pressures like antibiotics. One study 5 indicated viridans group streptococci individually responsible for 86% of 607 clinical isolates from endocarditis patients, of which 56% were resistant to penicillin G, a widely used antibiotic with previously high efficacy in the treatment of streptococcus bacteria (Bryskier, 2002). Patients at risk for infective endocarditis are given antibiotics, prescribed 1-hour before dental procedures
(American Heart Association, n.d.). When considering the protection provided by the biofilm, and the increase of resistance, the effectiveness of antibiotics alone is questionable.
Infective Endocarditis
Infective endocarditis is an infection of the endocardium, the innermost layer of the tissues which line the chambers of the heart. The infection of the endocardium is typically a result of streptococci, staphylococci, or fungus. The tricuspid valve is affected approximately 50% of the time, and less commonly the mitral and aortic valves are affected approximately 20% of the time. Endocarditis is not an easy or obvious diagnosis, and there are many clinical signs which can result in misdiagnosis. Surgery is required in approximately 60% of cases, and there are a large number of causative organisms; Streptococcus mutans is one of them (Niebauer, 2004).
Mycobacterium tuberculosis
Tuberculosis is a potentially severe infection that commonly attacks the lungs and can be spread through the air with coughing, sneezing, or talking.
Mycobacterium tuberculosis is the primary causative agent of Tuberculosis; humans are the single reservoir for the bacterium. Mycobacterium tuberculosis is 6 a Gram-positive bacterium, a member of the Mycobacteriaceae family, an abundant, nonmotile, rod-shaped bacterium, obligate aerobe, and a facultative intercellular parasite. The cell wall structure of M. tuberculosis is a crucial component to the virulence of the bacterium, made up of peptidoglycan and complex lipids containing mycolic acids, cord factor, and wax-D. These compounds assist in making the cell wall impermeable to stains or dyes and are resistant to many antibiotics, acids, alkaline compounds, osmotic lysis, and lethal oxidation.
Once a human host is infected with M. tuberculosis, the immune system responds by sending macrophages to surround the bacilli; a hard shell is then formed by the bacterium to keep the bacilli contained. The macrophage phagocytizes the hardened shell bacterium and remains un-activated, not destroying the bacterium. The unaffected bacterium multiplies within the macrophage, eventually bursting from the macrophage in much higher numbers and forming tubercles, which eventually cause caseous necrosis or death of lung cells. The formation of tubercles activates the host adaptive immune response.
Some of the macrophages become active at this stage and can destroy the bacterium, but many remain inactive or poorly activated, and the tubercles remain and continue to grow (Kenneth Todar, 2019). Recent research has indicated another evasion method used by M. tuberculosis. Upon phagocytosis by an active macrophage, M. tuberculosis secretes tuberculosis necrotizing toxin
(TNT), which hydrolyzes nicotinamide adenine dinucleotide (NAD+) in the macrophage; this results in death by necrosis for the macrophage and 7
Mycobacterium tuberculosis escapes containment evading adaptive immunity
(Sun J, 2015).
The length of treatment for M. tuberculosis is extensive, 6 - 30 months. Due to an increase in drug-resistant strains, many medications are given at once to treat it. Every drug prescribed for this bacterium is highly toxic to the liver and dangerous to the host (Mayo Clinic, 2019). Eradication of this disease will not be possible without a focus on new, faster, more effective treatment options.
Tuberculosis History
It is suspected that tuberculosis has been around for approximately three million years; it was named in 1834 by Johann Schonlein, and the discovery was announced in 1882 by Dr. Robert Koch. In 1882, tuberculosis (TB) killed one in every seven people infected. Due to the extensive history of tuberculosis, it has had many names, "phthisis" in ancient Greece, "tabes" in Rome, "schachepheth" in ancient Hebrew, "the white plague" in the 1700s, "consumption" in the 1800s, and "Captain of all these men death" in the 1800s. Before the discovery of a bacterial cause, TB was thought to be hereditary. In New England, it was believed that when a person died of TB, they came back as a vampire and infected the rest of the family. Throughout history, TB has continued to evade treatment and spread throughout the world. Today, TB is still the leading cause of death from bacterial infection. Testing for TB is performed through the injection. The injection contains a purified protein derivative of tuberculin and is placed under the skin of the patient. This injection triggers an immune response in 24-48 hours if TB antibodies are present. Radiographs, blood tests, and 8 interferon-gamma release assays can also confirm the presence of TB (Centers for Disease Control and Prevention, 2016).
Bacteriophage History
Bacteriophages were initially discovered by two scientists independently,
Frederick W. Twort in Great Britain (1915) and Felix d'Herelle in France (1917), it was Felix d'Herelle that coined the term bacteriophage – meaning bacteria eater, upon discovery.
The 1915 Twort discovery of bacteriophage occurred while trying to propagate another virus entirely, the vaccinia virus. Several bacterially contaminated plates had strange zones of clearing, and Twort realized that something, not visible by a microscope, was killing the bacteria. With limited time and resources, Twort proposed three potential reasons for these clearings: unusual activity of bacteria, an enzyme produced by bacteria, and or a tiny virus.
Twort published his findings and did not pursue research in this area (Keen,
2015).
Felix d'Herelle made a similar discovery when isolating bacteria from feces and urine in dysentery patients. This invisible microbe, according to d'Herelle, was only present in the absence of the pathogenic bacteria, Shiga bacillus was not found in other patients infected; also noting that this agent was host-specific, as it did not infect other strains of bacteria responsible for dysentery. At that time, d'Herelle referred to this invisible microbe as a "living-germ" (Norkin, 2015) and postulated that this antagonist microbe was an obligate bacteriophage not likely restricted to dysentery. Equipped with this research, d'Herelle began 9 making therapies using bacteriophage and successfully treated typhoid-infected chickens. Given the success in chickens, d'Herelle attempted human trials using fecal samples to identify the bacteria responsible for the infection and proliferated phage. To test for safety, d'Herelle ingested the phage. Once safety was confirmed, d'Herelle gave the patients the phage to ingest for treatment. After three successful treatments, phage therapy became a treatment for many individuals. The plague outbreak in 1920 and a cholera epidemic in 1927 were two times in history that phages were used successfully for the treatment of bacterial disease. Even with thousands of patients treated successfully, the lack of placebos discouraged the scientific community from considering phage- therapy useful. As such, interest in this treatment option diminished within the
United States and Western Europe. Eastern Europe and Russia, however, were cut off from western medicine, and interest in phage-therapy remained high.
Hundreds of papers were published, and many treatments using phages were developed. Phages, as a treatment for bacterial disease, are still commonly used there today (Norkin, 2015).
Not long after phage discovery, antibiotics were discovered and quickly became the go-to treatment for bacterial disease in many areas of the world, specifically those with exposure to western medicine. Though these bacterial viruses were discovered more than 100 years ago, the understanding of how a bacteriophage finds, adheres, and infects bacteria is still poorly understood.
Bacteriophage Diversity
10
Bacteriophages are obligate intracellular parasites that attack and lyse bacteria. Bacteriophages are the most abundant biological entity on earth with an estimation of 1031 phage particles, and enormous diversity. A bacteriophage is highly specific to its host and is more closely related to their host than another bacteriophage (Graham F. Hatfull, 2011). Bacteriophages actively engage in horizontal gene transfer and evolve with their bacterial counterparts, resulting in significant genetic diversity over billions of years. One 2018 study of comparative genomics indicated that 47.2% of mycobacteriophage genes and
58% of bacillus genes were unique, making relationships between phages hard to identify (Anh D. Ha, 2018). As of 2019, a phage database, PhagesDB.org, contained 17,625 individual phages across 13 different hosts. Of these phages, there are 127 different group allocations (clusters), one of which is a singleton, so unique it cannot be grouped with another phage (Daniel A Russell, 2019). The identified phages are only a small portion of phages that are on earth, and alone show how incredibly diverse this population is and how much more research needs to be done to understand diversity and co-relatedness.
Bacteriophage Anatomy and Lifecycles
Due to the high diversity of bacteriophages, bacterial virus taxonomy is ever-evolving. One method commonly used is an organization by the tail. As of
2009, the Order Caudovirales (Latin cauda “tail”) consisted of three primary families of tailed bacterial viruses: Myoviridae, Podoviridae, and Siphovirdae.
Myoviridae: long contractile tails, Podoviridae: short non-contractile tails, and 11
Siphovirdae: long non-contractile tails (Figure 1) (International Committee on
Taxonomy of Viruses, 2009).
A bacteriophage is very small, ranging from 20-200 nanometers. The basic anatomy is an icosahedral head, neck and collar, tail, sheath around the tail, tail fibers, and a base plate (Figure 2). The head stores the genetic material that is injected into the bacterial host; this can be DNA or RNA. The neck and collar are a stopper for the genetic material until the appropriate host has been found. The tail is a hollow tube for the injection of the genetic material, and sheath protein surrounding the tail is used to stabilize the phage on the surface of the bacteria. Tail fibers are receptors to identify the host and the baseplate at the bottom of the tail to bore into the bacterial cell wall and inject DNA/RNA. The phage uses its tail fibers to find its host, the base plate is employed to create a hole in the cell wall, the neck and collar release the genetic material from the head, and the tail injects the DNA like a syringe into the bacterium. Once the
DNA is in the bacterium, there are two lifecycles of bacteriophage, lytic and lysogenic (Figure 3).
In the lytic lifecycle, the phage attaches to the host cell and injects the
DNA. The phage DNA takes over the host machinery, new DNA and proteins are synthesized and assembled into virions; the cell lyses and releases the virions. Phages in this lifecycle are considered lytic or virulent phages and form clear plaques (Figure 4) (Gerald J. Tortora, 2013).
In the lysogenic lifecycle, the phage attaches to the host cell and injects the DNA. The phage DNA integrates into the host DNA and goes through cell 12 divisions with the bacterium. The inserted DNA into the host DNA is considered a prophage, and the bacterial cell is now a lysogen or temperate phage and forms cloudy plaques (Figure 5). Excision of the prophage or other environmental pressure can lead to the prophage becoming active and a switch to the lytic lifecycle for the phage. DNA and proteins are synthesized and assembled into virions; the cell lyses and releases the virions (Gerald J. Tortora, 2013).
Whether a phage is predisposed to these lifecycles or other environmental pressures result in these lifecycles is not well understood. Characterization of phage genomes to determine the genetic differences will play a key role in future understanding of lifecycle preference or determination.
Bacteriophage Specificity and Host Range
Bacteriophage specificity is the factor that results in the bacteriophage infecting one bacterium over another. The host range for a bacteriophage is the variation in bacterial hosts that one bacteriophage will infect.
Phages have incredible diversity, and some phages share little to no nucleotide sequences, making how they identify a host somewhat of a mystery.
Phages rarely infect hosts from other genera of bacteria, and species preferences within genera are shared among all phages. Some phages will only infect certain strains within a species, while other phages have been identified that will infect more than one species or strain within a genus. The Hatfull lab has postulated that mycobacteriophages host range is related to cluster designation and tail fiber genes. This suggests that mutations in the tail fiber genes result in the evolution of the bacteriophage that will allow infectivity in 13 other species and that relatedness of phage may be indicators for cross- infectivity (Deborah Jacobs-Sera a, 2012). Ultimately, there many factors to consider in the host range. Receptors, mutations in genes, transduction, and immunity to infection need to be identified to understand the host range fully.
Host range analysis in the lab, followed by genome sequencing, will provide a genomic comparison for factors of host range among bacteria, adding to the body of knowledge for how the host is determined for these bacteria.
Bacteriophage Treatment Today
Upon discovery of antibiotics, phage-therapy was largely abandoned in the United States and Western Europe. Advances in scientific understanding, new technology to study phage, an alarming increase of antibiotic resistance, and patient bacterial infections with complete resistance to all modern antibiotics, have brought the potential for phage-therapy back into the limelight.
In 2015, Tom Patterson, Ph.D., fell ill in Egypt while on vacation with his wife
Steffanie Strathdee, Ph.D. - the result of a deadly drug-resistant superbug
Acinetobacter baumannii – an Egyptian strain. Initially mistaken for food- poisoning, Tom was given antibiotics and sent back to his hotel. With no improvement from the antibiotics and his condition worsening, Tom was sent to
Germany for treatment. In Germany, it was determined that Tom had passed a gall stone that got lodged in a bile duct, which resulted in a cyst filled with this deadly bacterium. Medevac'd to San Diego, CA, for treatment, Tom was given many highly potent, but ultimately ineffective antibiotics. Tom had at least seven cases of septic shock, lost 100 pounds, and ended up in a coma. Tom's wife, a 14
Dean of Global Health, started to research alternative treatment options and found phage-therapy. Steffanie contacted phage researchers all over the United
States, and one responded. The causative bacteria were sent to this researcher, and it was tested against phage in the library and environmental sources; a match was found. Emergency approval from the Food and Drug Administration was given, and Tom was treated with the phages; he completely recovered.
Since that time, emergency approval for five patients, including Tom, have been approved and successful. Two years after Tom's case, in 2018, UC San Diego funded a three year 1.2 million dollar grant for the Center for Innovative Phage
Applications and Therapeutics (IPATH), for further advancement of phage research and therapeutic work (UC San Diego Health, 2019).
Some phage therapy for the food industry has been approved in the
United States. However, phage therapy for humans remains an unapproved treatment and requires emergency approval from the Food and Drug
Administration.
Lytic and Lysogenic Bacteriophages for Treatment of Infection
There is a renewed importance in studying bacteriophage for treatment of disease, and one key factor needs to be considered is lifecycle. The lifecycle of a phage, lytic or lysogenic, changes the way that the phage DNA interacts with a bacterial host.
The lysogenic lifecycle results in phage DNA integrating into the host
DNA, forming the prophage. This integration allows the bacteriophage to potentially share genes with the host, and create toxins from mutations in the 15 phage genome. This gene-sharing potential has been demonstrated for various toxins. Polymorphic toxins – multi-domain bacterial exotoxins, found in all major bacterial clades and demonstrate death or inhibition of competing bacteria, were found to frequently be a part of the DNA packaged by the bacteriophage (Anne
Jamet, 2019). Phage encoded genes that endure repeated replication and transcription after prophage activation can potentially play a role in toxin production; the diphtheria toxin of Corynebacterium diphtheriae is conjectured to be a result of phage-encoded genes (Patrick L. Wagner, 2002). Other examples are cholera toxin, Shiga-like toxin, botulinum toxin, and erythrogenic toxin – all found in bacteriophage genomes. Transfer of these toxins to harmless bacteria enhance virulence and increase the risk of disease (Nicol, 2003).
The lytic cycle lacks phage DNA integration and yet still raises concern for the host of the infectious bacteria. Gram-negative bacteria have an outer membrane of the cell wall, which contains a lipopolysaccharide layer (LPS); this layer has endotoxins (Figure 6). Biological activity of endotoxin is associated with Lipid A and immunogenicity, the provocation of the immune system, is associated with the polysaccharide components. In-vivo testing demonstrates that with the disintegration of the organism, endotoxin is released in high amounts (Figure 7). The quick-release of these endotoxins elicits an immune response (Kenneth Todar, Bacterial Endotoxin, 2012). Endotoxin is considered the most potent microbial mediator of disease and is associated with sepsis and septic shock (SM, 2007). 16
Treatment of bacterial disease using bacteriophage requires an understanding of the bacteria being treated. Treating Gram-negative bacteria with a lytic phage could result in sepsis from endotoxin release, and lysogenic phage could be more effective at slowly killing the bacteria with fewer side effects. However, treating bacteria without toxins, a lytic bacteriophage would be much efficient at clearing the infection quickly.
Summary
In this thesis, published methods for bacteriophage isolation of
Streptococcus specific phage were tested by researchers. Unsuccessful isolation of phage using those methods resulted in the revision of the methods as well as broadening the parameters of samples containing potential phage that infect Streptococcus species. Revision of methods did result in putative temperate phages.
Additionally, Mycobacterium smegmatis mc2155 bacteriophages that showed an ability to cross infect Mycobacterium tuberculosis H37Ra were tested for lysogen forming abilities. Bacteriophages that can form a lysogen bacterium are essential when considering phage as a potential treatment option over antibiotics. Therapeutic use of bacteriophages largely focuses on lytic bacteriophages, though lysogenic should also be researched. Identification of these bacteriophages allow researchers to study the genomic differences in bacteriophages and can further elucidate genes responsible for lifecycle that could be unique to this genera or species through comparison to lytic phages.
Additionally, the phages studied were able to cross-infect across species, further 17 genomic analyses could also be used to identify genetic differences in tail fibers that recognize host receptors through comparison to bacteriophages that do not cross-infect.
18
References American Heart Association. (n.d.). Infective Endocarditis. Retrieved from American Heart Association: https://www.heart.org/en/health-topics/infective- endocarditis Anh D. Ha, D. R. (2018). Comparative Genomic Analysis of 130 Bacteriophages Infecting Bacteria in the Genus Pseudomonas. Frontiers in Microbiology, 1-13. Anne Jamet, M. T.-G. (2019). A widespread family of polymorphic toxins encoded by temperate phages. BMC Biology, 75. Bryskier, A. (2002). Viridans group streptococci: a reservoir of resistant bacteria in oral cavities. Clinical Microbiology and Infection, 65-69. Centers for Disease Control and Prevention. (2016, December 12). Tuberculosis (TB). Retrieved from Centers for Disease Control and Prevention: https://www.cdc.gov/tb/worldtbday/history.htm Daniel A Russell, G. F. (2019, December 29). The Actinobacteriophage Database. Retrieved from The Actinobacteriophage Database: https://phagesdb.org Daniel Lopez, H. V. (2010). Biofilms. Cold Spring Harbor Perspectives in Biology, 1-12. David R. Harper, H. M. (2014). Bacteriophages and Biofilms. Antibiotics, 270-284. Deborah Jacobs-Sera a, L. b. (2012). On the nature of mycobacteriophage diversity and host preference. Virology, 187-201. Discovery and Development of Penicillin. (2019, December 26). Retrieved from American Chemical Society International Historic Chemical Landmarks. Discovery and Development of Penicillin: http://www.acs.org/content/acs/en/education/whatischemistry/landmarks/fle mingpenicillin.html Gerald J. Tortora, B. R. (2013). Microbiology An Introduction. Boston: Person. Graham F. Hatfull, R. W. (2011, October 1). Bacteriophages and their Genomes. Retrieved from NIH Public Access: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3199584/pdf/nihms308146.pdf International Committee on Taxonomy of Viruses. (2009, June). ICTV 9th Report (2011). Retrieved from International Committee on Taxonomy of Viruses: https://talk.ictvonline.org/ictv-reports/ictv_9th_report/dsdna-viruses- 2011/w/dsdna_viruses/67/caudovirales Keen, E. C. (2015). A century of phage research: Bacteriophages and the shaping of modern biology. HHS Public Access, 6-9. Kenneth Todar, P. (2012). Bacterial Endotoxin. Retrieved from Todar's Online Textbook of Bacteriology: http://textbookofbacteriology.net/endotoxin.html Kenneth Todar, P. (2019, December 27). Mycobacterium tuberculosis and Tuberculosis. Retrieved from Online Textbook of Bacteriology: http://textbookofbacteriology.net/tuberculosis.html Masatoshi Otsugu, R. N. (2017). Contribution of Streptococcus mutans Strains with Collagen-Binding Proteins in the Presence of Serum to the Pathogenesis of 19
Infective Endocarditis. American Society For Microbiology: Infection and Immunity, 1-17. Mayo Clinic. (2019, January 30). Tuberculosis. Retrieved from Mayo Clinic: Tuberculosis Neu, H. C. (1992). The Crisis in Antibiotic Resistance. Science, 1064-1073. Nicol, K. (2003, November 26). Virulence factors carried on Phage. Retrieved from Microbial Genetics: http://www.sci.sdsu.edu/~smaloy/MicrobialGenetics/topics/phage/phage- virulence.html Niebauer, E. A. (2004). Cardiology Explained. London: Remedica. Norkin, L. (2015, May 20). Felix d’Herelle, the Discovery of Bacteriophages, and Phage Therapy. Retrieved from Virology Molecular Biology and Pathogenesis: https://norkinvirology.wordpress.com/2015/05/20/felix-dherelle-the-discovery- of-bacteriophages-and-phage-therapy/ Oana Ciofu, E. R.-M. (2017). Antibiotic treatment of biofilm infections. APMIS, 304-319. Retrieved from Online Library Wiley: https://onlinelibrary.wiley.com/doi/epdf/10.1111/apm.12673 Patrick L. Wagner, M. K. (2002). Bacteriophage Control of Bacterial Virulence. American Society for Microbiology: Infection and Immunity, 3985-3993. SM, O. (2007). The host response to endotoxin, antilipopolysaccharide strategies, and the management of severe sepsis. PubMed, 365-377. Stephanie A. Fong, A. D.-J. (2017). Activity of Bacteriophages in Removing Biofilms of Pseudomonas aeruginosa Isolates from Chronic Rhinosinusitis Patients . frontiers in Cellular and Infection Microbiology, 417-418. Sun J, S. A. (2015, August 3). The tuberculosis necrotizing toxin kills macrophages by hydrolyzing NAD. Retrieved from PubMed: https://www.ncbi.nlm.nih.gov/pubmed/26237511 UC San Diego Health. (2019, December 29). Bacteriophage Therapy. Retrieved from UC San Diego Health: https://health.ucsd.edu/news/topics/phage- therapy/Pages/default.aspx World Health Organization. (2018, February 5). World Health Organization. Retrieved from Antibiotic resistance: https://www.who.int/news-room/fact- sheets/detail/antibiotic-resistance
20
Chapter 1 Figures
Figure 1: Bacteriophage Order Caudovirales. Myoviridae: long contractile tails, Podoviridae: short non-contractile tails, and Siphovirdae: long non-contractile tails https://www.pngwave.com/png-clip-art-wluvj
Figure 2: Bacteriophage Structure https://viralzone.expasy.org/resources/pro_VIRION_phage.jpg
Figure 3: Upon attachment and injection of genetic material the bacteriophage enters a lytic or lysogenic lifecycle. https://www.researchgate.net/post/please_mention_the_the_main_stages_of_a_phage_life_cycle _and_can_explain_the_major_difference_between_the_lytic_and_lysogenic_cycle
Figure 4: Presence of clear plaques on agar plates indicate a phage in a lytic lifecycle. http://www.sci.sdsu.edu/~smaloy/MicrobialGenetics/topics/phage/phage-lambda-plaque.gif 21
Figure 5: Presence of cloudy or turbid plaques on an agar plate indicate a phage in the lysogenic lifecycle.https://www.researchgate.net/profile/David_Hopwood/publication/18579974/figure/fig3/A S:601688721342478@1520465178735/Three-day-old-plaques-of-the-temperate-phage-VP5-on- S-coelicolor-A32-showing-a-clear.png
Figure 6: Components of lipopolysaccharide and other endotoxins http://1.bp.blogspot.com/_FN1wR3ASuIU/TB32KBUdvbI/AAAAAAAAB9Q/uODUTIKS9v8/s400/2.j pg
Figure 7: Gram-negative endotoxin release upon cell death. http://2.bp.blogspot.com/_FN1wR3ASuIU/SnXz_GvaoPI/AAAAAAAAAjg/i3q4J_dBqq4/s400/bacte rial+toxins.jpg
22
Chapter 2
Protocol Development for Bacteriophage Isolation using Streptococcus mutans
and Other Oral Bacteria as Hosts
This chapter is presented in the format of the article journal
23
Protocol Development for Bacteriophage Isolation for Streptococcus mutans and
Other Oral Bacteria
*Christine B. Emmons (corresponding author)
University of Nevada, Reno
Truckee Meadows Community College
7000 Dandini Blvd.
Reno, NV. 89512
(775) 673-7075
Laura A. Briggs
University of Nevada, Reno
Truckee Meadows Community College
(775) 673-7257
Frank Robertson
University of Nevada, Reno
Truckee Meadows Community College
(775) 420-9631
Running title: Protocol development of Streptococcus bacteriophages 24
Keywords: cross-infection, host range, Streptococcus mutans, Oral
bacteriophages
1
* This author is considered to be the first author 25
Abstract
Bacteriophages are important for maintaining the microbial balance of ecosystems and also are potential agents for therapeutics. Antibiotic resistance is compelling researchers to find more natural methods for treatment.
Streptococcus bacteria are contributors to dental carries and infective endocarditis. Their bacteriophages have largely evaded capture. Truckee
Meadows Community College has been isolating bacteriophage in conjunction with a national undergraduate research program since 2014. Researchers endeavored to create a protocol for successful capture and isolation of
Streptococcus bacteriophages. Lab strains of Streptococcus mutans and wild- type bacteria - cultured from the oral cavity, were analyzed as hosts. Saliva and soil were tested for the presence of bacteriophage. No novel phage was found.
However, procedural modifications resulted in putative temperate plaques, indicating potential isolation. Host range was analyzed using bacteriophages from host Mycobacterium smegmatis mc2155. Cross-infectivity was noted, infection rate was low. Environmental samples could prove beneficial in locating infective Streptococcus bacteriophage.
Introduction
Many bacteria share a common attribute, the ability to form a biofilm.
Biofilms can occur in almost any environment and are commonly found in the human body on teeth, skin, and the urinary tract. Formation of a biofilm provides protection for the bacteria, specifically, resistance to antimicrobials and protection 26 from host defenses 1. Tolerance mechanisms provided by the extracellular matrix is called phenotypic resistance. Phenotypic resistance can also lead to genetic resistance with antibiotic exposure via gene transfer and the biofilm allows this transfer to happen more effectively 2. Phenotypic and genetic resistance together with environmental pressures can result in bacteria within biofilms that cannot be treated with modern antibiotics. Streptococcus mutans is a meaningful contributor to dental caries, a breakdown of the teeth from acid- producing pathogenic bacteria. Streptococcus mutans are commonly found in the biofilm of the oral cavity of humans. While many viridans group streptococci constitute normal flora of the respiratory and gastrointestinal tract of humans,
Streptococcus mutans is a major pathogen and is also considered a causative agent of infective endocarditis. Entering the bloodstream from oral trauma such as dental procedures, daily oral care, or other injuries, Streptococcus mutans can wreak havoc on individuals with underlying heart conditions or who are at risk for infective endocarditis 3. Bacteriophages have been shown to reduce biofilm mass, penetrating the biofilm and effectively killing the bacteria within 4.
Bacteriophages are highly specific to their host and are more closely related to their host than another bacteriophage 5. Bacteriophages are very small, ranging from 20-200 nanometers and display two lifecycles after infection, lytic and lysogenic. In the lytic lifecycle the bacteriophage DNA takes over the host machinery, assembling virions until the cell lyses, thereby releasing the virions for further infection. Phages in this lifecycle are considered lytic or virulent phages and form clear plaques 6. Phages in the lysogenic lifecycle 27 integrate DNA into the host DNA and phage DNA goes through cell divisions with the bacterium. Excision of the prophage or other environmental pressure can lead to the prophage becoming active which catalyzes a switch to the lytic lifecycle for the phage. Excessive use of antibiotics has given rise to antimicrobial resistance 7. New resistance mechanisms are arising every day.
Previously treatable diseases like pneumonia, tuberculosis, and many others, are no longer treatable with antibiotics. While newly synthesized methods are being tested, the problem remains and other treatment options for bacterial infection and for disease need to be considered 8. The purpose of this study was to identify methods successful capture and isolation of Streptococcus bacteriophages.
Materials and Methods
1. Isolation of bacteriophage from salvia samples
S. mutans laboratory strains
Seven strains of S. mutans were obtained from Hardy Diagnostics (HD) and verified by Sanger DNA sequencing using 16S primers (See Bacterial species, and strain serotype verification and identification for methodology). These strains were cultured in vitro throughout the study (see culture conditions for culture methodology).
Isolation of bacteria from human saliva 28
Unfiltered human saliva was applied to 5% blood agar and brain heart infused agar (BHI) and incubated for 24 hours at 37°C in 5% CO2. Isolated colonies were purified using identical Streptococcus selective media under identical conditions.
Eleven unique colonies were cultured in vitro. (See culture conditions for culture methodology).
Bacterial species and strain serotype verification and identification.
DNA isolation and 16s rDNA amplification of eighteen bacterial strains was completed 9. Seven Streptococcus strains obtained from HD and one S. mutans strain (15), obtained from the APC Microbiome Ireland, University College Cork.
Ireland, and eleven bacterial specimens obtained from saliva were analyzed.
Primers Fn5 and Rn1 were used. DNA amplification was verified via agarose gel electrophoresis. The 16S PCR fragments were sequenced use
Sanger DNA sequencing at Nevada Genomics Center. Sequencing results were processed through NCBI BLAST for identification of the bacteria. See Table 1.
Bacteria culture conditions
All bacterial strains tested were grown in BHI broth incubated for 24 hours at
37°C in 5% CO2.
Double agar overlay spotting assay with BHI (DAOSABHI).
The presence of phage was examined via the DAOSABHI. BHI molten top agar with the addition of CaCl2 (10mM final concentration) was mixed with the 29 bacterial host, then applied to BHI solid agar. Five microliters of the unenriched saliva & enriched saliva were spotted on top of the BHI soft agar. After spot absorption, the plates were incubated for 24 hours at 37°C in 5% CO2. If no spots were identified the plates were incubated under the same conditions for seven days. A spot was classified as a circular clearing of bacteria at the location of the
5µl liquid application and was presumed to indicate bacteriophage infection.
Upon initial identification of spot production, the positive result was replicated in triplicate. Bacteriophage ØAPCMOL was used as a positive control for all S. mutans strains. There was no positive control for bacteria isolated from human saliva.
Phage capture by serial dilution with BHI (PCBSDBHI).
If a spot was observed as a result of the DAOSABHI and replicated in triplicate, the source (unenriched saliva & enriched saliva) of the potential bacteriophage infection was further characterized through a series of ten-fold serial dilutions.
Virus was not successfully harvested from spots. A liquid culture of the possible bacterial host was inoculated with 10µl of each serial dilution and then left undisturbed for 20 minutes. Following this infection time, the entirety of the infected liquid bacterial culture was mixed with molten BHI top agar and applied to the top of solid BHI agar in a petri dish and allowed to solidify. Plates were incubated for 24 hours at 37°C in 5% CO2. If no plaques were identified, the plates were incubated under the same conditions for seven days. A plaque was characterized as a clearing of bacterial growth which is suggestive of 30 bacteriophage infection and represents lysed bacterial cells. If plaques were noted, each isolated unique plaque in the most dilute plate was further purified in the same manner described until only one plaque morphology is observed. At this point the phage was considered pure and ready for further characterization.
2. Enrichment and isolation of bacteriophage using human saliva; Methods
1-4 (M1-M4)
Method 1-4
M1, Identification of phage infection using DAOSABHI: 4.5ml of human saliva was collected and centrifuged at 7168 xg for ten minutes. The supernatant was filter sterilized through a 0.45µm filter then a 0.22µm filter. 5µl of unenriched saliva was applied to ten of the bacterial hosts (1,2,7,11,12,13,14,15,17,18) via the DAOSABHI. The remaining saliva was enriched with each of the bacterial hosts using 0.4ml of filtered saliva + 0.4ml 2X BHI + 0.3ml host bacteria.
Enrichments were incubated for 24 hours at 37°C in 5% CO2. After incubation enrichments were centrifuged at 2000 xg for 10 minutes. The supernatant was filter sterilized using a 0.22µm filter. See Table 2 for the results.
M2 Identification of phage infection using DAOSABHI: Human saliva, 2.2ml, was collected. Centrifugation was withheld. The sediment within the saliva sample settled to the bottom of the conical tube. The supernatant was extracted, samples were filter sterilized through a 0.45µm filter then a 0.22µm filter. Five microliters of unenriched saliva was applied to six of the bacterial hosts 31
(13,14,15,16,17,18) via DAOSABHI. The remaining saliva was enriched with each of the bacterial hosts using 0.4ml of filtered saliva + 0.4ml 2X BHI + 0.3ml host bacteria. These enrichments were incubated for 24 hours at 37°C in 5%
CO2. The supernatant was removed, samples were filter sterilized using a
0.22µm filter. See Table 3 for results.
M3, Identification of phage infection using DAOSABHI: Seven milliliters of human saliva were collected on three consecutive mornings prior to oral care.
Sediment within the saliva sample was allowed to settle to the bottom of the conical tube. The supernatant was then extracted, filter sterilized through a
0.45µm filter then a 0.22µm filter. Five microliters of unenriched saliva was applied to ten of the bacterial hosts (11,12,13,14,15,16, 17,18, A, B) via
DAOSABHI. The remaining saliva was enriched with each of the bacterial hosts using 0.7ml of filtered saliva + 0.7ml 2X BHI + 0.5ml host bacteria + CaCl2 (10mM final concentration). Enrichments were incubated for 24 hours at 37°C in 5%
CO2. The supernatant was removed and filter sterilized using a 0.22µm filter. See
Table 4 for results.
M4: Nineteen milliliters of human saliva were collected on three consecutive mornings prior to oral care. The viscus mucous layer that was allowed to settle to the bottom. Three parallel saliva enrichments would be analyzed. The mucinous sediment at the bottom, unfiltered saliva, as well as filter-sterilized saliva were all enriched. The filtered saliva was first filtered sterilized through a 0.45µm filter 32 then a 0.22µm filter. The remaining mucinous sediment and unfiltered saliva were separated. All three were applied individually to the bacterial hosts using
0.46ml saliva + 0.8ml 2X BHI + 0.35ml host bacteria + CaCl2 (10mM final concentration) + 40% glycerol (final concentration). Enrichments were incubated for 24 hours at 37°C in 5% CO2. The supernatant was removed, and filter sterilized using a 0.22µm filter. See Table 5 for results.
Spot Isolation Verification
M3 Follow-up: Following the initial detection of the spot produced on (A) S. salivarius MitP4 from the enriched saliva, confirmation of the potential infection was performed in triplicate via DAOSABHI. After this assay, the isolation of the potential phage was attempted via PCBSDBHI. See Table 4 for results.
M4 Follow-up: Following the initial detection of spots produced on the (A) S. salivarius MitP4, (3) R. mucilaginosa E#9, (2) S. salivarius M17A10, (1) S. salivarius Mitp4 from enriched saliva, confirmation of the potential infection was performed in triplicate via DAOSABHI. After this assay, the isolation of the potential phage was attempted via PCBSDBHI. See Table 5 for results.
3. Enrichment of bacteriophage from soil samples
Bacteria Culture conditions 33
The bacteria used to enrich phage from soil samples were grown in BHI broth for
24 hours at 37°C in 5% CO2 conditions.
Enrichment Conditions
Thirty-six soil samples, numbered 1-36, taken from Reno, Nevada and the surrounding areas were utilized as a source for potential phage capture on the tested bacteria. One gram of soil was mixed with 6ml ddH2O + 1ml 10x BH + 1ml
3% AD supplement + CaCl2 (10mM final concentration) + 1ml host bacteria. The enrichment was incubated at 37°C in 5% CO2 conditions for seven days.
Following incubation enrichments were centrifuged at 2000 xg, and filter sterilized through a 0.22µm filter. Phage capture was then attempted via
DAOSPBHI.
Double agar overlay spotting assay with BHI (DAOSABHI)
The presence of phage was examined via the double agar overlay spotting assay
(DAOSABHI). BHI molten top agar with the addition of CaCl2 (10mM final concentration) was mixed with the bacterial host in question, then applied to the top of BHI solid agar. Five microliters of the filtered soil enrichments were then spotted on top of the BHI soft agar. After spot absorption, plates were incubated for 24 hours at 37°C in 5% CO2 conditions at 37°C. If no spots were identified, the plates were incubated under the same conditions for seven days. A spot was classified as a circular clearing of bacteria at the location of the 5µl liquid application and was presumed to indicate bacteriophage infection. Upon initial 34 identification of spot production, the positive result was replicated in triplicate
(See Table 8 for results). Following the replication of the results, phage capture would have been attempted via PCBSDBHI. Bacteriophage ØAPCMOL was used as a positive control for (15) S. mutans K51. There was no positive control for bacteria isolated from human saliva.
3. Host Range
Bacteria culture conditions
Liquid cultures of M. smegmatis mc2155 were incubated at 37°C for 24-48 hours, in a 7H9 broth, enriched with 3% AD (albumin dextrose) supplement, and CaCl2
(10mM final concentration). Other bacteria cross-tested were grown in BHI broth for 24 hours at 37°C in 5% CO2 conditions. See Table 6 for strain information and see Isolation of bacteria from human saliva for “saliva obtained bacteria” methodology.
Bacteriophage lysate conditions.
One-hundred-five phage lysates were filter sterilized through a 0.22µm filter, and infectivity verified via M. smegmatis mc2155 host. See DAOSA7H9 for methodology.
Double agar overlay spotting assay with 7H9 Media (DAOSA7H9) 35
The presence of phage infection was examined via DAOSA7H9. 7H9 molten top agar with the addition of CaCl2 (10mM final concentration) mixed with M. smegmatis, applied to the top of 7H9 sold agar and allowed to solidify. Five microliters of the bacteriophage liquid lysate was spotted on top of the 7H9 soft agar. After spot absorption, plates were incubated for 24-48 hours at 37°C. A spot was classified as a circular clearing of bacteria at the location of the 5µl liquid application and was presumed to indicate bacteriophage infection.
Mycobacteriophage Guilsminger was used as a positive control for M. smegmatis mc2155 infection.
Phage capture by serial dilution with BHI (PCBSDBHI)
Spots observed with DAOSABHI were replicated in triplicate, then further verified using ten-fold serial dilutions. A liquid culture of the bacterial host was inoculated with 10µl of each serial dilution and allowed to infect for 20 minutes. The infected liquid bacterial culture was mixed with molten BHI top agar and applied to the top of solid BHI agar and allowed to solidify. Plates were incubated for 24 hours at
37°C in 5% CO2. If no plaques were identified, the plates were incubated under the same conditions for an additional seven days. A plaque was characterized as a clearing of bacterial growth which is suggestive of bacteriophage infection and represents lysed bacterial cells. If plaques were noted, virus was isolated from a unique plaque on the most dilute plate and further purified in the same manner described until only one plaque morphology was observed. At this point the 36 phage was considered pure, and ready for further characterization. See Table 7 for results.
Results
Six Streptococcus species were identified in saliva. No bacteriophage were captured using the initial protocol. Spots were detected on S. salivarius MitP4
(A) with the addition of CaCl2, virus could not be harvested from the spots. The enrichment protocol with the addition of glycerol produced spots for S. salivarius
MitP4 (A), S. salivarius MitP4 (1), R. mucilaginosa, and S. salivarius M17A10.
Virus could not be harvested from spots. Host range results (Table 7) indicated infectivity by phage for hosts S. salivarius (A), Strep spp. (B), S. salivarius (1), R. mucilaginosa, and bacterial clone (9). Virus could not be harvested from spots; efficiency of plating was zero. Soil enrichment produced spots on host R. mucilaginosa in triplicate. See Tables for additional results.
Discussion
Bacteriophages have characteristics that make them potential therapeutic agents for bacterial infection. They are highly specific, effectively kill targeted pathogenic bacteria, and are safe for the human host. Streptococcus bacteria display as much as 56% resistance to some antibiotics, bacteriophage as a potential treatment option is not only significant but important as the rates of resistance continue to rise. The isolation from oral samples needs further elucidation. 37
Putative infection was discovered when samples were enriched in media containing CaCl2 (10mM final concentration), glycerol (40% final concentration), and without centrifugation. Results indicate that the bacteriophages are more delicate than other bacteriophages when compared to hardier
Mycobacteriophages found in soil. Calcium chloride assists the bacteriophage by making the bacterial host cell more competent thereby more accessible to foreign DNA 10; thus, making infection easier. Glycerol further improved the isolation of bacteriophage. Glycerol stabilizes bacteriophage by reducing protein flexibility and mimicking their natural environment. Cross-infection of bacteriophage was not effective, though cross-infection was noted, though noted the infection rate was not sufficient to be considered for therapeutic use, efficiency of plating equaled zero. Soil enrichment produced putative phage for
Rothia mucilaginosa, an opportunistic oral bacterial pathogen, this was not pursued, yet indicates soil as a potential medium for the isolation of bacteriophage for oral bacteria.
Further research using the addition of CaCl2, glycerol, termination of centrifugation, and enrichment protocols could be advantageous for bacteriophage isolation with a saliva medium. Future purification of soil bacteriophages against Streptococcus mutans and other oral species to confirm the bacteriophage captured have an efficiency of plating of 10-3 or better to be 38 considered for further characterization. This study shows that environmental samples have the potential for capturing oral bacteriophage for oral pathogens.
39
Acknowledgments
We thank Truckee Meadows Community College and the University of Nevada,
Reno provided lab resources and academic and faculty support. Funding was provided by Nevada INBRE NSF #1118679. A generous gift of the positive control for Streptococcus mutans was provided by APC Microbiome Ireland,
University College Cork. Ireland. Some phages were obtained from the
University of Pittsburg, Hatfull Lab.
40
Authorship Confirmation Statement:
Christine B Emmons, Frank Robertson, Laura A Briggs:
All authors have met the requirements listed below. The in-lab work was completed by Christine and Frank. All designs completed by Christine and
Laura. Analysis, interpretation, and review of submissions have been completed by all authors.
Substantial contributions to the conception or design of the work; or the acquisition, analysis, or interpretation of data for the work; and drafting the work or revising it critically for important intellectual content; and final approval of the version to be published; and agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.
41
The authors have no competing financial interests.
42
References
1. Daniel Lopez, H. V. (2010). Biofilms. Cold Spring Harbor Perspectives in Biology, 1-12. 2. Oana Ciofu, E. R.-M. (2017). Antibiotic treatment of biofilm infections. APMIS, 304-319. Retrieved from Online Library Wiley: https://onlinelibrary.wiley.com/doi/epdf/10.1111/apm.12673 3. Masatoshi Otsugu, R. N. (2017). Contribution of Streptococcus mutans Strains with Collagen-Binding Proteins in the Presence of Serum to the Pathogenesis of Infective Endocarditis. American Society For Microbiology: Infection and Immunity, 1-17. 4. Stephanie A. Fong, A. D.-J. (2017). Activity of Bacteriophages in Removing Biofilms of Pseudomonas aeruginosa Isolates from Chronic Rhinosinusitis Patients. frontiers in Cellular and Infection Microbiology, 417-418. 5. Graham F. Hatfull, R. W. (2011, October 1). Bacteriophages and their Genomes. Retrieved from NIH Public Access: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3199584/pdf/nihms308146. pdf 6. Gerald J. Tortora, B. R. (2013). Microbiology An Introduction. Boston: Person. 7. Neu, H. C. (1992). The Crisis in Antibiotic Resistance. Science, 1064-1073. 8. World Health Organization. (2018, February 5). World Health Organization. Retrieved from Antibiotic resistance: https://www.who.int/news-room/fact- sheets/detail/antibiotic-resistance 9. Barghouthi, S. A. (2011). A Universal Method for the Identification of Bacteria Based on General PCR Primers. Indian J Microbiol, 430-444. 10. Azka Asif, H. M. (2017). Revisiting the Mechanisms Involved in Calcium Chloride Induced Bacterial Transformation. Frontiers in Microbiology, 2169.
43
Chapter 2 Tables
Saliva obtained bacteria (HD) obtained bacteria 1. Streptococcus. salivarius 11. Streptococcus mutans Chdc Ym62 Mitp4 2. Streptococcus. salivarius 12. Streptococcus mutans Chdc Ym62 M17A10 3. Rothia mucilaginosa E#9 13. Streptococcus mutans K51 4. Neisseria mucosa DNF00552 14. Streptococcus mutans gene Jcm 5175 5. Neisseria perflava 0023-01R 15. Streptococcus mutans K51 (Ireland) 7. Streptococcus spp. Firmi-9 16. Streptococcus mutans BM29 8. Streptococcus sanguinis K47 17. Streptococcus ferus gene 9. Uncultured bacterial clone. 18. Streptococcus mutans Jmc 5175 10. Neisseria flavescens 8642 (A) Streptococcus salivarius MitP4 (B) Streptococcus spp. Table 1: NCBI BLAST results for all bacteria tested with this project.
Saliva Spot y/n Spot y/n HD obtained Spot y/n Spot y/n obtained Nonenriched enriched bacteria Nonenriched enriched bacteria saliva saliva saliva saliva 1. S. n n 11. S. mutans n n salivarius Chdc Ym62 Mitp4 2. S. n n 12. S. mutans n n salivarius Chdc Ym62 M17A10 7. n n 13. S. mutans n n Streptococcus K51 spp Firmi9 14. S. mutans n n gene Jcm5175 15. S. mutans n n K51 (Ireland) 16. S. mutans n n BM29 17. S. ferus n n gene 18. S. mutans n n Jmc 5175
Table 2: Results of original protocol for phage capture via human saliva enrichment.
44
HD obtained bacteria Spot y/n Nonenriched Spot y/n enriched saliva saliva 13. S. mutans K51 n n 14. S. mutans gene Jcm5175 n n 15. S. mutans K51 (Ireland) n n 16. S. mutans BM29 n n 17. S. ferus gene n n 18. S. mutans Jmc 5175 n n Table 3: Method 2 Results (Termination of Centrifugation) for phage capture via human saliva enrichment.
Saliva Spot y/n Spot y/n HD obtained Spot y/n Spot y/n obtained Nonenriched enriched bacteria Nonenriched enriched bacteria saliva saliva saliva saliva (A) S. n y 11. S. mutans Chdc n n salivarius Ym62 MitP4 (B) Strep n n 12. S. mutans Chdc n n spp. Ym62 13. S. mutans K51 n n 14. S. mutans gene n n Jcm5175 15. S. mutans K51 n n (Ireland) 16. S. mutans BM29 n n 17. S. ferus gene n n 18. S. mutans Jmc n n 5175 Table 4: Method 3 results (Addition of CaCl2 & Continuous Saliva Collection) for phage capture via human saliva enrichment.
45
Enriched bacteria for Spot Enriched bacteria for Spot Enriched bacteria Spot mucinous sediment y/n filtered saliva y/n for non-filtered y/n saliva
(A) S. salivarius Y (A) S. salivarius MitP4 Y (A) S. salivarius Y MitP4 MitP4 (B) Strep spp. N (B) Strep spp. N (B) Strep spp. N 1. S. salivarius Mitp4 N 1. S. salivarius Mitp4 N 1. S. salivarius Y Mitp4 2. S. salivarius N 2. S. salivarius M17A10 Y 2. S. salivarius Y M17A10 M17A10 3. R. mucilaginosa E#9 Y 3. R. mucilaginosa E#9 Y 3. R. mucilaginosa Y E#9 7. Streptococcus spp. N 7. Streptococcus spp. N 7. Streptococcus N Firmi-9 Firmi-9 spp. Firmi-9 9. Uncultured bacterial N 9. Uncultured bacterial N 9. Uncultured N clone. clone. bacterial clone. 11. S. mutans Chdc N 11. S. mutans Chdc Ym62 N 11. S. mutans Chdc N Ym62 Ym62 12. S. mutans Chdc N 15. S. mutans K51 N 12. S. mutans Chdc N Ym62 (Ireland) Ym62 13. S. mutans K51 N 16. S. mutans BM29 N 13. S. mutans K51 N 14. S. mutans gene Jcm N 17. S. ferus gene N 14. S. mutans gene N 5175 Jcm 5175 15. S. mutans N 18. S. mutans Jmc 5175 N 15. S. mutans K51 N K51(Ireland) (Ireland) 16. S. mutans BM29 N 16. S. mutans N BM29 17. S. ferus gene N 17. S. ferus gene N 18. S. mutans Jmc 5175 N 18. S. mutans Jmc N 5175 Table 5: Method 4 results (Addition of glycerol) for phage capture via human saliva enrichment.
Saliva obtained bacteria 1. S. salivarius Mitp4 2. S. salivarius M17A10 3. R. mucilaginosa E#9 7. Streptococcus spp. Firmi-9 9. Uncultured bacterial clone. (A) S. salivarius MitP4 (B) Strep spp. Table 6: Potential bacterial hosts tested during cross infectivity assay
46
S. salivarius (A) Strep spp. (B) S. salivarius (1) R. mucilaginosa (3) bacterial clone (9)
Tony Tony Kayla Perez Tony #74 Rose Tony Kayla Rose DeeCee Kayla Table 7: Results of host range assay of phage that spotted in triplicate. Phage host listed on the top line.
# Spot X3 on Spot X3 on Spot X3 # Spot X3 on Spot X3 Spot X3 (A)Salivariu (3) R. on (15) S. (A)Salivariu on (3) R. on (15) S. s MitP4 mucilaginos mutans s MitP4 mucilagin mutans (Y/N) a E#9 (Y/N) K51 (Y/N) osa E#9 K51 (Y/N) (Y/N) (Y/N) 1 N Y N 19 N Y N 2 N N N 20 N N N 3 N N N 21 N N N 4 N N N 22 N N N 5 N Y N 23 N N N 6 N Y N 24 N Y N 7 N Y N 25 N Y N 8 N N N 26 N N N 9 N Y N 27 N N N 10 N N N 28 N N N 11 N N N 29 N N N 12 N N N 30 N N N 13 N N N 31 N N N 14 N N N 32 N N N 15 N N N 33 N N N 16 N Y N 34 N Y N 17 N Y N 35 N N N 18 N N N 36 N N N Table 8: Results for the Bacteriophage capture via soil enrichment.
47
Chapter 3
Cross Infection, Host Range, and Lysogeny of Mycobacterium smegmatis
mc2155 bacteriophages against Mycobacterium tuberculosis H37Ra
This chapter is presented in the format of the article journal
48
Cross Infection, Host Range, and Lysogeny of Mycobacterium smegmatis
mc2155 bacteriophages against Mycobacterium tuberculosis H37Ra
*Christine B. Emmons (corresponding author)
University of Nevada, Reno
Truckee Meadows Community College
7000 Dandini Blvd.
Reno, NV. 89512
(775) 673-7075
*Tina Slowan-Pomeroy (corresponding author)
University of Nevada, Reno
Truckee Meadows Community College
7000 Dandini Blvd.
Reno, NV. 89512
(775) 674-7916
Laura A. Briggs
University of Nevada, Reno 49
Truckee Meadows Community College
(775) 673-7257
Frank Robertson
University of Nevada, Reno
Truckee Meadows Community College
(775) 420-9631
Running title: Cross infect, host range, lysogeny, TB phages
Keywords: cross-infection, host range, lysogeny, TB phages, Mycobacterium
2
* These authors contributed equally to this work and are considered to be co-first authors 50
Abstract
Today, tuberculosis (TB) is still the leading cause of death from bacterial infection. Due to an increase in drug-resistant strains, many medications are given at once to treat it, each highly toxic to the liver and dangerous to the host.
Bacteriophages are natural predators of this bacterium and are a possible therapeutic agent to battle the resistance and dangers of medications to the patient. This research analyzed the cross-infection of bacteriophages, to the original host, Mycobacterium smegmatis mc2155, and new host Mycobacterium tuberculosis H37Ra. Of one-hundred and five mycobacteriophages tested, seven displayed cross-infection capabilities, and two displayed lysogenic properties. Lysogen-forming bacteria play an essential role in the use of bacteriophage as treatment options. Integration into the genome allows for the potential transfer of toxic genes. Future studies characterizing the genes displayed in these lysogen-formers will allow better understanding of the genes responsible for lifecycle preference.
Introduction
On average, phages range in size from 20-200 nanometers (nm) and comprise a myriad of morphologies. However, all consist of a capsid ("head") that contains genetic material (usually dsDNA) and a tail, which allows the Phage to attach to the bacterial host and inject its genetic material. The process of a bacteriophage injecting its unique DNA into the host, replicating within the 51 bacterium using the host's replication machinery and lysing the cell, is known as the lytic cycle. This presents as clear areas, called plaques, on a bacterial lawn.
Bacteriophage can also enter a lysogenic, or temperate, cycle whereby phage
DNA integrates into bacterial host DNA remaining dormant in the cell. This lifecycle presents as turbid, or cloudy, plaques. A lysogen is defined as a bacterium that contains a phage genome. A prophage refers to the genomic material of the phage incorporated into the genome of the host bacterium 1. The lifecycle displayed is the result of the expression of many different genes, some identified, and many not.
The lysis cassette contains endolysin genes responsible for the lysis of the bacterial host and includes lysin A, lysin B, and Holin. Lysins A and B are generally esterase’s, glycosidases, amidases, or peptidases and are involved in hydrolyzing the mycolic acids and peptidoglycan layers of the mycobacterial host cell wall 2,3. Holin proteins regulate when lysis of the bacterial host occurs by puncturing the cell membrane from within, creating a membrane-spanning channel, and allowing endolysins to reach the cell wall for destruction 3,4. The immunity cassette refers to the phage genes involved in lysogeny, defined as the integration of the phage genome into the bacterial host genome, and includes proteins such as integrase, excisionase, CRO, and immunity repressors 5.
Integrase is a recombinase responsible for the integration of the phage genome into the host genome and excision out of it, while excisionase aids integrase in this process 6,7. Cro and other immunity repressor genes prevent transcription of lytic genes, thereby maintaining lysogeny 7,8. Immunity repressors also prevent 52 superinfection, defined as an event where multiple phages infect a single bacterial host. Typically lysogens (bacteria containing a prophage) are immune to superinfection by similar phages via repressor-mediated superinfection immunity
1,7. Whether a phage is predisposed to these lifecycles or if other environmental pressures play a role is not well understood. Characterization of phage genomes to determine the genetic differences will play a key role in future understanding of lifecycle preference or determination.
Bacteriophage specificity is the factor that results in the bacteriophage infecting one bacterium over another. Host range for a bacteriophage is the variation in bacterial hosts. Both specificity and range require more research due to the incredible diversity in bacteriophages. Some phages share little to no nucleotide sequences, making how they identify a host somewhat of a mystery.
Phages rarely infect hosts from other genera of bacteria, and species preferences within genera are shared among all phages. Some phages will only infect certain strains within a species, while other phages will infect more than one species or strain within a genus. The Hatfull lab has correlated mycobacteriophages host range to cluster designation and tail fiber genes. This suggests that mutations in the tail fiber genes result in the evolution of the bacteriophage that allows infectivity in other species and that relatedness of phage may be indicators for cross-infectivity 9. Ultimately, there are many factors to consider for host range preferences, such as receptors being identified, mutations in genes, and immunity. 53
The two most common methods for measuring host range are spot testing and plaquing. Spot testing, a small volume of phage spotted onto a bacterial lawn, is a simple way to rapidly screen for phage infection. However, due to bacterial lysis without phage infection, this technique can result in false positives and overestimates the host range of a particular phage 10,11. Lysis without phage infection can occur when a large number of phage attach to the bacterial cell wall, overwhelm it, and lyse it. This results in residual lysins or holins in the phage lysate that lyse the bacteria 10,11. The plaquing method, whereby phage infection is determined by the formation of plaques on a bacterial lawn, can be a more accurate measure of phage infection. However, not all hosts are as receptive to plaquing even if there is successful phage infection 11.
With a renewed importance in studying bacteriophage for treatment of disease, one key factor needs to be considered, lifecycle. The lifecycle of a phage, lytic versus lysogenic, changes the way that the phage DNA interacts with a bacterial host. The lysogenic lifecycle results in phage DNA integrating into the host DNA, forming the prophage. This integration allows the bacteriophage to potentially share genes with the host, potentially creating toxins from mutations to the phage genome. This gene-sharing potential has been demonstrated for various toxins. Polymorphic toxins – multi-domain bacterial exotoxins, found in all major bacterial clades and demonstrate death or inhibition of competing bacteria, were found to frequently be a part of the DNA packaged by the bacteriophage 12. Phage encoded genes that endure repeated replication and transcription after prophage activation can potentially play a role in toxin 54 production; the diphtheria toxin of Corynebacterium diphtheriae is conjectured to be a result of phage-encoded genes 13. Other examples are cholerae toxin,
Shiga-like toxin, botulinum toxin, and erythrogenic toxin – all found in bacteriophage genomes. Transfer of these toxins to harmless bacteria enhance virulence and increase the risk of disease 14. The lytic cycle lacks phage DNA integration, yet still raises concern for the host of the infectious bacteria. Gram- negative bacteria have an outer membrane of the cell wall, which contains a lipopolysaccharide layer - endotoxins. The biological activity of endotoxin is associated with Lipid A. Immunogenicity, or the provocation of the immune system, is associated with the polysaccharide components 15. In-vivo testing demonstrates that with the disintegration of the organism, endotoxin is released in high amounts. The quick release of these endotoxins elicits an immune response 16. Endotoxin is considered the most potent microbial mediator of disease and is associated with sepsis and septic shock 17.
Treatment of bacterial disease using bacteriophage requires an understanding of the bacteria being treated, treating Gram-negative bacteria with a lytic phage could result in sepsis from endotoxin release, and lysogenic phage could be more effective at slowly killing the bacteria with fewer side effects.
However, when treating bacteria without toxins, a lytic bacteriophage would be much faster at clearing the infection and eliminating potential toxic gene transfer.
Tuberculosis is a potentially severe infection that commonly attacks the lungs and can be spread through the air with coughing, sneezing, or talking.
Mycobacterium tuberculosis, a Gram-positive bacterium, is the primary causative 55 agent of tuberculosis; humans are the single reservoir for the bacterium. Today,
TB is still the leading cause of death from bacterial infection. The length of treatment for M. tuberculosis is extensive, 6 - 30 months. Due to an increase in drug-resistant strains, many medications are given at once to treat an infection.
Every drug prescribed for this bacterium is highly toxic to the liver and dangerous to the host 18. Eradication of this disease will not be possible without a focus on new, faster, less harmful treatment options.
Materials and methods
Bacteria culture conditions
Cultures of M. smegmatis mc2155 were incubated at 37°C for 24-48 hours in a
Middlebrook 7H9 broth, enriched with 3% Albumin Dextrose (AD) supplement,
CaCl2 (10mM final concentration) or plated on Luria Agar (LA). Cultures of M. tuberculosis H37Ra were first cultivated on Lowenstein-Jensen slanted medium, incubated at 37°C for 2-3 weeks. Cultures were grown in liquid medium containing Middlebrook 7H9 broth with Albumin Dextrose Catalase (ADC) supplement under the same incubation conditions. Mycobacterium tuberculosis
H37Ra was plated on Middlebrook 7H10 agar with Middlebrook OACD (Oleic
Albumin Dextrose Catalase) supplement.
Bacteriophage lysate conditions. 56
Phage lysates were filter sterilized through a 0.22µm filter, and infectivity verified via the original host M. smegmatis mc2155 host. See DAOSA for the methodology.
Double agar overlay spotting assay (DAOSA)
The presence of phage infection was examined via DAOSA. Middlebrook 7H9 molten top agar with the addition of CaCl2 (10mM final concentration) was mixed with M. smegmatis mc2155 and M. tuberculosis H37Ra, respectively, then applied to the top of LA (M. smegmatis mc2155) or 7H10 with OADC (M. tuberculosis H37Ra) solid agar and allowed to solidify. Five microliters of phage lysate was spotted on inoculated Middlebrook 7H9 soft agar. After spot absorption, the plates were incubated for 24-48 hours or 2-3 weeks for M. smegmatis mc2155 and M. tuberculosis H37Ra, respectively, under aerobic conditions at 37°C. A spot was classified as a circular clearing of bacteria at the location of the 5µl liquid application and was presumed to indicate bacteriophage infection. Mycobacteriophage Guillsminger was used as a positive control for M. smegmatis mc2155 infection. There was no positive control for M. tuberculosis
H37Ra. All phage lysates capable of producing a spot for M. tuberculosis H37Ra were tested in triplicate 19.
Bacteriophage Titer & Acquisition of Efficiency of Plating Assay (EOP).
Bacteriophage lysates titer was calculated by the equation listed below. 57
( ) (DF) = Viral Titer ( )
A plaque was characterized as a clearing of bacterial growth, which is suggestive of bacteriophage infection, representing lysed bacterial cells, and the presence of virions. Ten-fold serial dilutions of phage lysates were used to infect a liquid culture of M. smegmatis mc2155 and M. tuberculosis H37Ra for 20 minutes.
Following this infection time, the entirety of the infected liquid bacterial culture was mixed with molten Middlebrook 7H9 top agar and applied to solid LA (M. smegmatis mc2155) and 7H10 with OADC (M. tuberculosis H37Ra) and incubated under standard conditions. Dilution plates with 20-200 plaques were used for EOP determination. The EOP assay compares the infection rate of the phage on its original host, M. smegmatis mc2155, to that of M. tuberculosis
H37Ra (equation below).
. = EOP .
An EOP equal to one means the phage infects M. tuberculosis H37Ra at the same frequency as M. smegmatis mc2155. An EOP of zero indicates the phage infected M. smegmatis mc2155, but not M. tuberculosis H37Ra. An EOP greater than zero suggests the phage infects M. tuberculosis H37Ra with less efficiency then M. smegmatis mc2155. An EOP of at least 10-3 indicates possible therapeutic use of the phage. See Figures 1, 2, and Table 1.
Bacteriophage Lysogeny Assay
58
Mesa Production by Serial Dilution
Phages that were capable of infecting M. tuberculosis H37Ra in triplicate was further examined through a ten-fold serial dilution (100-10-6). Five microliters of each diluted lysate was spotted on 7H10 with OADC plates using DAOSA.
However, after incubation, (See culture conditions), "Mesa" spots were looked for at the location of each 5µl dilution spot. A "Mesa" spot was classified as a circular clearing of bacteria at the location of the 5µl liquid application with a very cloudy center, and represents bacterial growth in the presence of phage, and provides a source for potential lysogens. The entirety of the bacteriophage lysogeny assay was also run with M. smegmatis mc2155 in parallel as a control for each mycobacteriophage. See Table 2.
Lysogen Candidate Production
The center of the M. tuberculosis H37Ra Mesa spots produced from each phage lysate was streaked on 7H10 with OADC plates and incubated in standard conditions. Six well isolated bacterial colonies were chosen as potential lysogens and further examined via the patch test assay. See Table 2.
Putative Lysogen Patch Test Assay & Subsequent Lysogen purification.
Each of the six isolated bacterial colonies produced from the above procedure were patched on two 7H10 with OADC plates. Two plates were create using host
M. tuberculosis H37Ra: One plate without Middlebrook 7H9 molten top agar and one plate with the inoculated Middlebrook 7H9 molten top agar. If the bacterial 59 patch shed phage and therefore produced a clearing of lysis around its periphery, the corresponding bacterial batch on the plate without inoculated top agar was utilized and streaked in triplicate for purification. Upon purification, this assay was run once more. If the bacteria were able to produce a clearing of lysis once more, a viable putative lysogen was captured and stored for further studies. See Table
2.
Results
Of 105 mycobacteriophage lysates tested for host specificity, 20 (19%) infected
M. tuberculosis H37Ra. Plaque testing results from the 20 phages capable of infecting M. tuberculosis H37Ra indicate that some phages are more successful in the infection process than others. While none of the 20 phages tested were capable of infecting M. tuberculosis H37Ra at the same frequency as M. smegmatis mc2155, several (30%) were able to do so within an efficiency threshold of 10-3: Peachy (EOP: 2.01E-2), Guilian2 (7.68E-3), OldHouse (EOP:
3.34E-3), Erimy (EOP: 2.55E-3), ScoobyBlue (2.05E-3), and Warrosco (EOP:
1.89E-3). An efficiency threshold of 10-3 suggests the phage is able to cross- infect. Several other phages (20%) were still able to infect M. tuberculosis
H37Ra, but at a reduced rate: Guillsminger (EOP: 3.53E-4), Alishanda (EOP:
1.76E-5), Lazanducci (EOP: 6.02E-7), and Zose (EOP: 9.97E-8). Phages not capable of infecting M. tuberculosis H37Ra via the plaquing method were:
Battleborn, Chandler, ChandlerG, DeeCee, Gronion, Idlewild, IdlewildPark,
Manaxia, Mistake, and Sierra15. 60
Bacteriophage with successful plaquing were verified via the spot method. The
10 bacteriophage tested were: Peachy (EOP: 2.01E-2), Guilian2 (7.68E-3),
OldHouse (EOP: 3.34E-3), Erimy (EOP: 2.55E-3), ScoobyBlue (2.05E-3), and
Warrosco (EOP: 1.89E-3 Guillsminger (EOP: 3.53E-4), Alishanda (EOP: 1.76E-
5), Lazanducci (EOP: 6.02E-7), and Zose (EOP: 9.97E-8). Of the ten bacteriophages tested, seven spotted on both M. tuberculosis H37Ra and M. smegmatis mc2155 (Figure 1). These seven remaining bacteriophages were used in a lysogeny experiment; two of which formed potential lysogens on both
M. tuberculosis H37Ra and M. smegmatis mc2155. See figures 1 and 2. See
Tables 1 and 2.
Discussion
The objective of this study was to identify Mycobacteriophages capable of cross- infection and lysogen formation. Bacteriophages obtained from Mycobacterium smegmatis mc2155 as a host were tested for cross-infection of Mycobacterium tuberculosis H37Ra. The bacteriophages that displayed successful cross- infection were then tested to determine lysogen formation. Therapeutic use of bacteriophages is focused primarily on lytic bacteriophages. While lytic bacteriophages are important when considering phages as a treatment option, lysogenic bacteriophages are also important. Lysogenic bacteriophages display a unique lifecycle that slows down the process of killing bacteria; this could be beneficial when treating bacteria such as Gram-negative bacteria. Additionally, these bacteriophages can be used in genomic studies to elucidate genes that are 61 responsible for lifecycle that may be unique to this genus, species, or strain.
Additional genomic studies could use these bacteriophages to characterize receptors or attachment differences when compared to bacteriophages that do not display an ability to cross-infect across strain. Lysogeny confirmation requires two steps: mesa production and bacterial clearing. The production of a mesa represents cell growth in the presence of bacteriophage. The bacterial clearing shows that phage is present in the cells cultured over the bacterial lawn.
Both of these steps indicate bacteriophage within the bacterial cells is a lysogen.
The final steps are verification of spontaneous phage release and efficacy of lysogeny. Scooby Blue and Old House were the two bacteriophages that created lysogens in M. tuberculosis H37Ra. Three bacteriophages in this study, Erimy,
Guillian 2, and Guillsminger, did produce mesa’s but did not display phage release when patched on a host bacteria plate. This could be due to the stability of the lysogen; thus, the conditions of this procedure did not induce the excision of the genome. Therefore, all five of the bacteriophages that displayed a mesa spot should be considered in additional lysogen studies. The EOP that for each of the potential lysogens was 10-4 or better, indicating that all five of the aforementioned bacteriophages infect the new host with good efficiency.
Verification of spontaneous phage release and efficacy of lysogeny was not tested in this protocol.
Analysis of the seven phages capable of cross infecting M. tuberculosis H37Ra is ongoing. Cluster designation for six of these phage genomes sequenced to date 62 indicates that all are members of Cluster A or K. This is in keeping with previous research performed in the Hatfull lab on host range analysis 20.
Future Directions
Genomes of phages capable of infecting M. tuberculosis H37Ra could be compared to phage genomes incapable of infecting M. tuberculosis H37Ra in order to determine genetic commonalities and differences. Additionally, phages that formed lysogens could be verified genetically and their genomes compared to lytic phages. Genome comparative analysis using bioinformatics would aid in the determination of genes in common in an effort to search for putative genes involved in cross infectivity as well as lysogeny and lytic lifecycle. Future work may involve the knockout of putative proteins to determine involvement in cross infection and lifecycle. This study will contribute to current studies in addressing the mechanisms of host specificity and lysogeny.
63
Acknowledgments
Truckee Meadows Community College and the University of Nevada, Reno, provided lab resources and academic and faculty support. Funding was provided by Nevada INBRE NSF #1118679. Some phages were obtained from the
University of Pittsburg, Hatfull Lab.
64
Authorship Confirmation Statement:
Christine B Emmons and Tina Slowan-Pomeroy (Co-first authors), Frank
Robertson (Second Author), Laura A Briggs (Third Author):
Substantial contributions to the conception or design of the work; or the acquisition, analysis, or interpretation of data for the work; and drafting the work or revising it critically for important intellectual content; and final approval of the version to be published; and agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.
65
The authors have no competing financial interests.
66
References 1. Dedrick R. M., J.-S. D.-B. (2016). Prophage-mediated defense against viral attack and viral counter-defense. Nature Microbiology, 16251(2). 2. Hatfull, G. F. (2010). Mycobacteriophages: genes and genomes. Annu, Rev. Microbiol, 64, 331-356. 3. Payne, K. a. (2012). Mycobacteriophage endolysins: diverse and modular enzymes with multiple catalytic activities. PLoS ONE, 7(3). 4. Phagesdb. (n.d.). Retrieved from phagesdb.org 5. Pedulla, M. e. (2003). Origins of highly mosaic mycobacteriophage genomes. Cell, 113, 171-182. 6. Cho, E. H. (2002). Interactions between integrase and excisionase in the phage lambda excisive nucleoprotein complex. Journal of Bacteriology, 184(18), 5200-5203. 7. Mediavilla, J. J. (2000). Genome Organization and characterization of mycobacteriophage Bxb1. Molecular Microbiology, 38(5), 955-970. 8. Takeda, Y. F. (1977). Cro Regulatory Protein specified by bacteriophage lambda. Journal of Biological Chemistry, 252(17), 6177-6183. 9. Deborah Jacobs-Sera a, L. b. (2012). On the nature of mycobacteriophage diversity and host preference. Virology, 187-201. 10. Potera, C. (2013). Phage Renaissance - New Hope Against Antibiotic Resistance. Environmental Health Perspective, 121(2). 11. Ross, A. W. (2016). More Is Better: Selecting for Broad Host Range Bacteriophages. Frontiers in Microbiology, 7(1352). 12. Anne Jamet, M. T.-G. (2019). A widespread family of polymorphic toxins encoded by temperate phages. BMC Biology, 75 13. Patrick L. Wagner, M. K. (2002). Bacteriophage Control of Bacterial Virulence. American Society for Microbiology: Infection and Immunity, 3985-3993. 14. Nicol, K. (2003, November 26). Virulence factors carried on Phage. Retrieved from Microbial Genetics: http://www.sci.sdsu.edu/~smaloy/MicrobialGenetics/topics/phage/phage- virulence.html 15. Christian Alexander, E. T. (2001). Bacterial lipopolysaccharides and innate immunity. Journal of Endotoxin Research, 167-202 16. Kenneth Todar, P. (2012). Bacterial Endotoxin. Retrieved from Todar's Online Textbook of Bacteriology: http://textbookofbacteriology.net/endotoxin.html 17. SM, O. (2007). The host response to endotoxin, antilipopolysaccharide strategies, and the management of severe sepsis. PubMed, 365-377. 18. Mayo Clinic. (2019, January 30). Tuberculosis. Retrieved from Tuberculosis: https://www.mayoclinic.org/diseases- conditions/tuberculosis/diagnosis-treatment/drc-20351256 19. Slowan-Pomeroy, T. (2018, December). Phage Diversity of Six Mycobacteriophages and Host Range of 105 Mycobacteriophages in Four Mycobacteria Including Mycobacterium Tuberculosis H37Ra. 67
20. Pope, W. H.-S. (2015, April 28). Whole genome comparison of a large collection of mycobacteriophages reveals a continuum of phage genetic diversity. Retrieved from eLife: https://doi.org/10.7554/eLife.06416
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Chapter 3 Figures
Comparison of phage titer (PFU/mL) on M. smegmatis and M. tuberculosis
1.00E+10 1.00E+08 1.00E+06 1.00E+04 Phage Phage Titer 1.00E+02 1.00E+00
Erimy Zose Peachy Chandler DeeCee GronionGuilian2 Idlewild ManaxiaMistake Sierra15 AlishandaBattleborn ChandlerG Lazanducci OldHouse Warrosco Guillsminger IdlewildPark ScoobyBlue
M. smegmatis Titer (PFU/mL) M. tuberculosis Titer (PFU/mL)
Figure 1:8Comparison of phage titer on M. smegmatis and M. tuberculosis. Phage titer on M. smegmatis provides a baseline from which to compare phage infection efficiency on M. tuberculosis.
69
Phage EOP on M. smegmatis compared to M. tuberculosis 1.00E+00 Zose Erimy Peachy 1.00E-01 Guilian2 Alishanda Warrosco OldHouse Lazanducci ScoobyBlue Guillsminger
1.00E-02 2.01E-02 7.68E-03 2.55E-03 3.34E-03 1.00E-03 2.05E-03 1.89E-03
3.53E-04 1.00E-04 Phage EOP
1.00E-05 1.76E-05
1.00E-06
6.02E-07 1.00E-07 9.97E-08
1.00E-08
Figure 2:9Phage Efficiency of Plating (EOP) on M. smegmatis compared to M. tuberculosis. Of the 20 phages plaque tested for infection efficiency, 30% were capable of infecting M. tuberculosis within an efficiency threshold of 10-3: Peachy, Guilian2, OldHouse, Erimy, ScoobyBlue, and Warrosco; 20% were able to infect M. tuberculosis at a reduced rate: Guillsminger, Alishanda, Lazanducci, and Zose. Of the 20 phages tested for infection efficiency, 50% were not able to infect M. tuberculosis via the plaquing method: Battleborn, Chandler, ChandlerG, DeeCee, Idlewild, IdlewildPark, Manaxia, Mistake, and Sierra15.
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Chapter 3 Tables
M. smegmatis M. tuberculosis Phage EOP Titer (PFU/mL) Titer (PFU/mL) Alishanda 4.11E+09 7.22E+04 1.76E-05 Battleborn 4.11E+09 0 0 Chandler 7.67E+09 0 0 ChandlerG 1.56E+10 0 0 DeeCee 2.00E+03 0 0 Erimy 8.22E+08 2.10E+06 2.55E-03 Gronion 4.22E+09 0 0 Guilian2 7.22E+08 5.55E+06 7.68E-03 Guillsminger 3.00E+10 1.06E+07 3.53E-04 Idlewild 1.50E+10 0 0 IdlewildPark 1.97E+10 0 0 Lazanducci 6.44E+08 3.88E+02 6.02E-07 Manaxia 6.22E+09 0 0 Mistake 4.60E+10 0 0 OldHouse 2.33E+09 7.80E+06 3.34E-03 Peachy 1.04E+10 2.10E+08 2.01E-02 ScoobyBlue 1.47E+09 3.00E+06 2.05E-03 Sierra15 2.44E+10 0 0 Warrosco 5.11E+09 9.66E+06 1.89E-03 Zose 2.78E+09 2.77E+02 9.97E-08 Table 1:9Efficiency of Plating (EOP) for all mycobacteriophages tested that can cross infect M. tuberculosis compared to the original host, M. smegmatis.
71
Procedure Completed Guills *Scooby Guillian ming *Old Alisha Lazan Warr Blue Peachy Erimy 2 er House nda ducci osco Zose M. smegmatis spot + + + + + + + + + + M. tuberculosis + spot + + + + + + (lytic) Produced a mesa MS + + + + + + Produced a mesa on TB + + + + + Spot on bacteria on a bacterial - clearing MS + + Spot on bacteria on a bacterial - clearing TB + +
Table 2:10Ten bacteriophage with cross infection capabilities tested for lysogeny. Scooby Blue and Old House bacteriophages spotted, produced mesas, and bacterial clearings on both M. tuberculosis and original host bacteria M. smegmatis making 2 of the 10 tested (20%) potential lysogens for M. tuberculosis and M. smegmatis. • *Indicates potential lysogen candidate
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Chapter 4 Conclusions / Summary
73
Conclusions
Bacteriophages have characteristics that make them potential therapeutic agents for bacterial infection. They are highly specific, effectively kill targeted pathogenic bacteria, and are safe for the human host. Streptococcus bacteria display as much as 56% resistance to some antibiotics, bacteriophage as a potential treatment option is not only significant but important as the rates of resistance continue to rise. The isolation from oral samples needs further elucidation.
Putative infection was discovered when samples were enriched in media containing CaCl2 (10mM final concentration), glycerol (40% final concentration), and without centrifugation. Results indicate that the bacteriophages are more delicate than other bacteriophages. Calcium chloride assists the bacteriophage by weakening the cell wall of the host bacteria, making infection easier. Glycerol further improved the isolation of bacteriophage. Glycerol stabilizes bacteriophage by reducing protein flexibility and mimicking their natural environment. Cross-infection of bacteriophage was not effective, though cross- infection was noted, though noted the infection rate was not sufficient to be considered for therapeutic use, efficiency of plating equaled zero. Soil enrichment produced putative phage for Rothia mucilaginosa, an opportunistic oral bacterial pathogen, this was not pursued, yet indicates soil as a potential medium for the isolation of bacteriophage for oral bacteria. 74
Further research using the addition of CaCl2, glycerol, termination of centrifugation, and enrichment protocols could be advantageous for bacteriophage isolation with a saliva medium. Future purification of soil bacteriophages against Streptococcus mutans and other oral species to confirm the bacteriophage captured have an efficiency of plating of 10-3 or better to be considered for further characterization. This study shows that environmental samples have the potential for capturing oral bacteriophage for oral pathogens.
Of 105 mycobacteriophage lysates tested for host specificity, 20 (19%) infected M. tuberculosis H37Ra. Plaque testing results from the 20 phages capable of infecting M. tuberculosis H37Ra indicate that some phages are more successful in the infection process than others. While none of the 20 phages tested were capable of infecting M. tuberculosis H37Ra at the same frequency as
M. smegmatis mc2155, several (30%) were able to do so within an efficiency threshold of 10-3: Peachy (EOP: 2.01E-2), Guilian2 (7.68E-3), OldHouse (EOP:
3.34E-3), Erimy (EOP: 2.55E-3), ScoobyBlue (2.05E-3), and Warrosco (EOP:
1.89E-3). An efficiency threshold of 10-3 suggests the phage is able to cross- infect. Several other phages (20%) were still able to infect M. tuberculosis
H37Ra, but at a reduced rate: Guillsminger (EOP: 3.53E-4), Alishanda (EOP:
1.76E-5), Lazanducci (EOP: 6.02E-7), and Zose (EOP: 9.97E-8). Phages not capable of infecting M. tuberculosis H37Ra via the plaquing method were: 75
Battleborn, Chandler, ChandlerG, DeeCee, Gronion, Idlewild, IdlewildPark,
Manaxia, Mistake, and Sierra15.
Bacteriophage with successful plaquing were verified via the spot method.
The 10 bacteriophage tested were: Peachy (EOP: 2.01E-2), Guilian2 (7.68E-3),
OldHouse (EOP: 3.34E-3), Erimy (EOP: 2.55E-3), ScoobyBlue (2.05E-3), and
Warrosco (EOP: 1.89E-3 Guillsminger (EOP: 3.53E-4), Alishanda (EOP: 1.76E-
5), Lazanducci (EOP: 6.02E-7), and Zose (EOP: 9.97E-8). Of the ten bacteriophages tested, seven spotted on both M. tuberculosis H37Ra and M. smegmatis mc2155. These seven remaining bacteriophages were used in a lysogeny experiment; two of which formed potential lysogens on both M. tuberculosis H37Ra and M. smegmatis mc2155. The objective of this study was to create a lysogen with each bacteriophage. Pre-verification requires two steps: mesa production and bacterial clearing. The production of a mesa represents cell growth in the presence of bacteriophage. The bacterial clearing shows that phage is present in the cells cultured over the bacterial lawn. Both of these steps indicate bacteriophage within the bacterial cells is a lysogen. The final steps are verification of spontaneous phage release and efficacy of lysogeny. Scooby Blue and Old House were the two bacteriophages that created lysogens in M. tuberculosis H37Ra and M. smegmatis mc2155. Verification of spontaneous phage release and efficacy of lysogeny was not tested in this protocol. 76
Analysis of the seven phages capable of cross infecting M. tuberculosis
H37Ra is ongoing. Cluster designation for six of these phage genomes sequenced to date indicates that all are members of Cluster A or K. This is in keeping with previous research performed in the Hatfull lab on host range analysis.
Future Directions
Genomes of phages capable of infecting M. tuberculosis H37Ra could be compared to phage genomes incapable of infecting M. tuberculosis H37Ra in order to determine genetic commonalities and differences. Additionally, phages that formed lysogens could be verified genetically and their genomes compared to lytic phages. Genome comparative analysis using bioinformatics would aid in the determination of genes in common in an effort to search for putative genes involved in cross infectivity as well as lysogeny and lytic lifecycle. Future work may involve the knockout of putative proteins to determine involvement in cross infection and lifecycle. This study will contribute to current studies in addressing the mechanisms of host specificity and lysogeny.
In the lysogeny study, two protocols were not completed; spontaneous phage release and efficacy of lysogeny. These verifications were not completed due to financial limitations and the extended growth time for M. tuberculosis; both protocols should be completed to verify the lysogeny of bacteriophages.