ABSTRACT
“The Induction, Purification and Host Range of 4 Lysogenic Staphylococcus aureus
Bacteriophages”
Carlo Manzana
Director: Tamarah Adair, Ph.D.
This research examined a possible correlation between bacteriophage resistance
of Staphylococcus aureus and the presence of integrase genes. Four lysogenic
Staphylococcus aureus bacteriophages were isolated and their lytic ability was
determined. Phages were obtained through Mitomycin C phage induction and verified
through positive spot tests on a panel of 10 different isolates. Phages were then assayed
for their host range and compared to a well-known S. aureus phage, Phage K. The 4
induced phages exhibited less lytic activity than Phage K. Moreover, the results suggest
that there is no clear correlation between phage resistance and the type or number of
prophage integrase genes present in the host bacteria.
APPROVED BY DIRECTOR OF HONORS THESIS:
________________________________________________
Dr. Tamarah Adair, Department of Biology, Baylor University
APPROVED BY THE HONORS PROGRAM:
________________________________________________
Dr. Andrew Wisely, Director
DATE: _____________________________
THE INDUCTION, PURIFICATION AND HOST RANGE OF FOUR LYSOGENIC
STAPHYLOCOCCUS AUREUS BACTERIOPHAGES
A Thesis Submitted to the Faculty of
Baylor University
In Partial Fulfillment of the Requirements for the
Honors Program
By
Carlo Manzana
Waco, Texas
December 2012
i
TABLE OF CONTENTS
Chapter One: Review of Literature
1
Chapter Two: Introduction
8
Chapter Three: Methods and Materials
13
Chapter Four: Results
18
Chapter Five: Conclusion and Discussion
26
Appendices
32
Work Cited
39
ii
PREFACE
Of the multitudes of bacteria combated by mankind, one species has been known
to be resistant to many antimicrobial treatments. Equipped with the necessary genes to
evade antimicrobial compounds, this species has proved to be a pathogen that is difficult
to eliminate. Its prominence in toxicity, pathogenicity, and adaptability has popularized it
as the renowned “Superbug”: Staphylococcus aureus.
iii
ACKNOWLEDGEMENTS
Special thanks to Dr. Tamarah Adair, who was an incredible source for guidance and
knowledge throughout the duration of this project. Her passion for teaching and research
inspired me to be diligent in my endeavors and produce the highest quality work. I
sincerely thank her for the opportunity to work on this project and for her help in seeing
the completion of this research and the improvement of my scholasticism.
I would also like to thank the Baylor Honors Program, the Baylor Honors College, and
the Baylor Biology department for funding my research and the conference presentations
associated with my research.
Lastly, I would like to thank my thesis defense committee, consisting of Dr. Tamarah
Adair, Dr. Walter Holmes, and Dr. Marcie Moehnke, who took the time to read, analyze,
and critique my thesis.
iv
CHAPTER ONE
Review of Literature
Characteristics of Staphylococcus aureus
Staphylococcus aureus has unique characteristics that allow for identification.
With the naked eye, S. aureus colonies can be easily recognized by their golden pigment.
When Gram-stained and viewed under a compound microscope at 1000 magnification,
these colonies appear to be small cocci grouped in grape-like clumps. Experiments also
screen for the presence of mannitol fermentation and coagulase activity to verify for S.
aureus. Testing positive for catalase activity also helps differentiate the Staphylococcus
genera from other gram-positive cocci (Bergey’s Manual, 1994).
Epidemiology
Staphylococcus aureus can exist in locations that have high salt concentrations. In
fact, numerous studies have shown S. aureus to reside in the anterior nares of humans
without posing any immediate harm to its host. Studies show that 18-33% of people
living in the United States are nasal carriers of this organism (Mainous et al. 2006; Bode
et al. 2010; Rohde et al. 2009). More significant is the nasal carriage rate of Methicillin
Resistant S. aureus (MRSA). Methicillin is a potent semi-synthetic antibiotic derived
from the beta-lactam penicillin. The carriage rate for MRSA amounts to 2-3% nationally
(Mainous et al. 2006). However, in clinical or hospital settings, the incidence of MRSA
rises to 6% nationally (Eveillard et al. 2004). Recent studies have shown that the
1
prevalence of MRSA can be even greater. In a twelve year study following drug resistant
bacteria among older patients in a hospital, 56.8% of all recovered cultures were resistant
to methicillin (Denkinger et al. 2012). The percentage of MRSA strains in that hospital
rose from 41% in 1998 to 56.8% in 2009. The high incidence of S. aureus nasal carriage
has been known to correlate to the superbug’s enhanced epidemiology and pathogenicity.
Though nasal carriage does not directly cause illness to the host organism, it does
present itself to be a risk factor in acquiring staph infections. The relationship between
nasal carriage and infection is exemplified best in post-surgical complications, whereby
patients harboring S. aureus in their anterior nares before operations are more likely to be
infected than patients without nasal colonization (Gupta et al. 2011; Weinstein, 1959;
Williams et al. 1959). Moreover, studies have shown that the rate of S. aureus nasal
carriage is directly proportional to the incidence of bacteremia (Von Eiff et al. 2001) and
in-hospital infections (Davis et al. 2004).
Staphylococcus aureus Infections and Disease
Staphylococcus aureus can perturb the regular functions of the human body in a
variety of ways. The most common entrances for infection are the skin and soft tissues.
The resulting infection is usually accompanied by an abscess or pus. There are a myriad
of diseases caused by S. aureus. Necrotizing fasciitis, (Miller et al. 2005) myositis and
pyomyositis (Pannaraj et al. 2006) are some examples of the severe outcomes of S.
aureus infections. Recently, there has been an increase in the incidence of these types of
staph infections (Dalager-Pedersen et al. 2011). Research indicates that a major risk
2
factor is invasive operative procedures that require debridement (Sreeramoju et al. 2010).
Though S. aureus infections are usually found in soft tissues and skin, it is by no means
the only area of infection.
Another common entrance route is through the gastrointestinal tract. There have
been many outbreaks of food poisoning due to ingestion of S. aureus with active
enterotoxin genes (Martín et al. 2004). Contaminated foods usually results from
displacing bacteria from their contaminated handlers directly onto the food (Argudin et
al. 2010). The most frequent food poisoning outbreaks have resulted from ingesting
contaminated dairy and meat products (Tamarapu et al. 2001; Wieneke et al. 1993)
Though most cases do not necessitate hospitalization, patients will usually experience
“nausea, violent vomiting, abdominal cramping, with or without diarrhea” (Argudin et al.
2010).
Staphylococcus aureus can also cause infections in other regions. Common
infections include urinary tract infections (Baba-Moussa et al. 2007), necrotizing
pneumonia (Mongkolrattanothai et al. 2003) and osteomyelitis (Mantadakis et al. 2012).
A rare, but more severe, infection caused by S. aureus occurs when it enters the cranial
area and causes meningitis (Huang et al. 2010). These infections are difficult to treat,
because of the lack of access to the cranial region. That is not to say, though, that other
forms of staph infections can be easily treated. On the contrary, S. aureus possesses a
variety of characteristics that make it elusive from anti-microbial agents.
3
Pathogenicity
The pathogenicity of S. aureus is caused by two main components: toxins and
proteins. These two secretory products function to not only protect the bacterium from an
immune response, but also to transform host tissues into nutrients used for rapid
reproduction and growth. Alpha-hemolysin (Hla) has been known to interact with the
phospholipids of membranes to disrupt tissue function by lysing cells (Wiseman, 1975).
In small quantities, Hla can also trigger apoptosis (Bantel et al. 2001). The lysis of
erythrocytes, in particular, further enhances the pathogenicity of S. aureus by releasing
iron nutrients for the bacteria to absorb (Andreini et al. 2008). Iron is an essential nutrient
for multiple intracellular redox reactions. Therefore, iron starvation is lethal to S. aureus.
As a result, the ability to acquire iron from the hemoglobin of human red blood cells
allows S. aureus to continue to replicate and infect nearby tissues (Pishchany et al. 2010).
Other notable toxins include pyrogenic superantigens and leukocidins. Pyrogenic
superantigens consist of two main toxins: enterotoxins and toxic shock syndrome toxins.
Enterotoxins are emetic agents usually released in the gastrointestinal tract of humans and
cause vomiting and diarrhea (Casman, 1965). Acting as superantigens, toxic shock
syndrome toxins cause severe hypotension to the host organism. These symptoms are
caused by the increase of cytokines. Other symptoms, such as rash, fever and organ
failure, may also occur (Larkin et al. 2009). Both enterotoxins and toxic shock syndrome
toxins are classified as pyrogenic superantigens because of their ability to induce fevers
and prompt an extreme immune system response (Marrack and Kappler, 1990).
Leukocidins, on the other hand, increase the virulence of S. aureus by lysing white blood
4
cells and necrotizing local tissues (Lina et al. 1999). The action of these leukocidins
makes S. aureus nearly impossible to kill by immune system responses.
The pathogenicity of S. aureus is further enhanced by the arsenal of polypeptides
that also gives the bacterium the ability to evade or disrupt antibacterial and immune
system agents. One of the most significant of these is Protein A. The cell wall of every S.
aureus bacterium is covered with this antigen-like protein, which sequesters and disables
a prominent antibody, immunoglobulin G, by binding to its fragment crystallizable region
and making the variable region point away from the targeted bacterium (Jensen, 2007).
The deactivation of immunoglobulin G permits bacterial cells to persist inside the host
and continue to infect the surrounding tissues.
Another protein that contributes to pathogenicity is coagulase. This enzyme is one
of the hallmarks of S. aureus. The gene for coagulase is found on the agr locus, which is
the same as that for Protein A (Lebeau et al. 1994). With respect to pathogenicity, the
coagulase enzymes provide a wall of fibrin clots that render it difficult for phagocytes to
destroy the bacteria (Zajdel et al. 1973). Early studies on coagulase confirmed that S.
aureus strains that have inactive coagulase are less pathogenic than those that do
(Chapman et al. 1934). Coagulase is now used as a required gene for S. aureus
identification.
Though Protein A and coagulase increase the virulence of S. aureus, the most
significant peptides produced by S. aureus are arguably those that contribute to the
bacteria’s resistance to antibiotics.
5
Antibiotic Resistance
Equipped with genes that render it to be protected from immune systems, S.
aureus has had a considerable advantage over its host. With the discovery of penicillin by
Alexander Fleming in 1928, the means to cure staph infections became possible
(Fleming, 1928). Since then, numerous other antibiotics were either discovered or
synthesized. Unfortunately, antibiotics have only been a temporal solution.
The overuse of penicillin quickly led to the emergence of antibiotic resistant S.
aureus strains that dominate hospital populations (Jawetz, 1963). Antibiotics that
significantly reduced staph infections in the mid-1900s were becoming less and less
effective as the century progressed (Parker, 1964). As new antibiotics were introduced,
similar patterns emerged. The Centers for Disease Control and Prevention performed a
study from 1992 to 2004 on nosocomial infections in Intensive Care Units (ICU) and
determined that nationally, 59.5% of all S. aureus nosocomial infections from the ICU
were caused by a methicillin resistant strain. Moreover, the study found that the presence
of MRSA strains increased 11% from 1998 to 2003 (National Nosocomial Infections
Surveillance System 2004).
Over the past few decades, methicillin resistant S. aureus strains have become
more prevalent. The mecA gene is the gene responsible for providing resistance to
methicillin. It functions by producing Penicillin Binding Protein 2a (PBP 2a), a version of
Penicillin Binding Protein (PBP). The mechanism of action for methicillin is to bind to
PBP which halts cell wall synthesis. PBP 2a circumvents methicillin by binding to it less
than its counterpart, PBP (Hartman and Tomasz, 1984). The mec gene is located on a
6
SCC, a Staphylococcal cassette chromosome. Several versions of this gene have been
identified and sequenced (Turlej et al. 2011).
Researchers have been using the SCC and mec types to classify the types of
MRSA. To date, eleven SCCmec types were discovered, with some that have subtypes
(Turlej et al. 2011). SCCs contribute to the increasing incidence of MRSA because they
allow easy trading of genetic elements through horizontal gene transfer (Ma et al. 2001).
Originally, MRSA was classified as either hospital or community acquired and these
categories were associated with specific mecA types. As horizontal gene transfer occurs
between these two populations, the line is beginning to blur (Huang et al. 2006). The rise
of antibiotic resistance has made it clear that reliance on antibiotics alone will not be
sufficient to fight Staphylococcus aureus.
7
CHAPTER TWO
Introduction
History of Bacteriophage Therapy
The advent of multidrug resistant bacteria has influenced the exploration of new
therapeutic avenues against S. aureus. One of the leading treatment options is
bacteriophage therapy. Bacteriophages are viruses that have the ability to infect and kill
bacteria. While bacteriophages were not officially discovered until 1917 by Felix
D’Herelle, their presence was known over two decades prior. In 1896, Ernest Hankin
became the first scientist to encounter these antibacterial agents, and in 1915 Frederick
Twort found similar antibacterial activity against Bacillus subtilis (Hankin 1896, Twort
1915).
However, these antimicrobials were not utilized nor coined until Felix D’Herelle
isolated bacteriophages from fecal matter and treated French soldiers for dysentery
during World War I (Summers 1999). D’Herelle heavily supported the hypothesis that
these invisible bacteria killers were viruses and provided a detailed account on the
methods to isolate them. Before bacteriophage therapy could fully develop as a viable
antibacterial treatment, though, the discovery of penicillin by Alexander Fleming rapidly
overshadowed bacteriophages as the main medicine against bacteria. As a result,
bacteriophages were neglected by most Western pharmaceutical companies and research
groups through the twentieth century.
8
In Eastern Europe, however, phage therapy has continuously been in use and has
been under medical experimentation (Miedzybrodzk et al. 2007). Research institutions,
such as the Eliava Institute in Georgia and the Hirszfeld Institute in Poland, have been
advancing bacteriophage therapy by developing phage treatments against an assortment
of different bacterial species, including Staphylococcus aureus (Sulakvelidze et al. 2001;
Slopek et al. 1987).
The emergence of antibiotic resistant bacteria, however, has rekindled interest in
bacteriophage therapy in the Western hemisphere. Its main advantage over antibiotics is
the high specificity for a particular host species, which allow them to effectively attach to
their target cells without risking unintended lysing of other species (Gu et al. 2011).
There have been several studies on the viability of bacteriophages as therapeutic
agents. Cocktail solutions have been produced on a small-scale level and have been
successfully tested on burn victims (Merabishvilli et al. 2009). The holin group, the group
of proteins responsible for lysing bacterial cell walls, has also been studied and developed
into recombinant lysins. Research groups have determined that recombinant lysins have
synergistic effects with antibiotics in treating against S. aureus infections (Daniel et al.
2010) and can act independently to eliminate S. aureus colonization (Fenton et al. 2010).
Resistance to Bacteriophages
The main question linked to bacteriophage therapy is the possibility of host
resistance to bacteriophages. Resistance rates can vary dramatically depending on the
bacteriophage examined. For example, susceptibility studies have shown that S. aureus
9
phage Stau2 can lyse 80% of tested S. aureus isolates while S. aureus phage K can lyse
only 47% (Hsieh et al. 2011; O’Flaherty et al. 2005).
The methods of phage resistance have been studied thoroughly but are far from
being completely understood. Bacteria have always had methods of preventing phage
infection. These processes can occur on the cell wall receptors of the bacteria or inside
the cell. Researchers have classified resistance to bacteriophages into four categories:
adsorptive resistance, phage-genome uptake blocks, abortive resistance and restriction
modification (Hyman and Abedon, 2010). Each mechanism of resistance blocks the
phage from infecting the bacteria. Adsorptive resistance occurs with incompatible phage
and host bacterial receptors. Phage-genome uptake blocks include the existence of a
similar prophage preventing infection. Restriction and abortive blocks include restriction
enzymes, CRISPR genes and gene expression blocks. Each of these resistance pathways
have been described fully in Hyman and Abedon, 2010.
Obtaining Bacteriophages
One of the main factors in choosing a bacteriophage as a treatment option is the
host range, or the number of different strains a phage can lyse. Research groups have
isolated bacteriophages from areas such as sewage, endotracheal tubes, and milk in
search for the most lytic phage (Synott et al. 2009, Hsieh et al. 2011, Garcia et al. 2009).
Isolation of lytic bacteriophages from the environment is not the only method for
obtaining a phage. Bacteriophages can be induced from their lysogenic state to the lytic
state by stressing the bacteria. Stress can be applied through the addition of Mitomycin C
to the liquid culture or through illumination with ultraviolet light. Induced phages can
10
then be used as lytic phages against other strains (Hoshiba et al. 2010).
Characterization of Bacteriophages
While bacteriophages have been known to the scientific community for almost a
century (de Gialluly et al. 2003), a uniform method for simple phage characterization and
classification has not yet been produced. Though bacterial strains have been classified
based on the types of phages harbored, the opposite method of characterization is more
difficult. Moreover, identifying phages by morphological family type is also difficult as it
requires the accumulation of a highly concentrated solution of phage for transmission
electron microscopy.
In an attempt to categorize S. aureus phages, Goerke et al. correlated the integrase
genes of S. aureus prophages to virulence genes, specifically those that coded for PantonValentine Leukocidin and different enterotoxins, and suggested categorizing S. aureus
prophages by screening for the presence of these genes (2009). The seven most common
integrases genes were determined and labeled as Sa1int, Sa2int, Sa3int, Sa4int, Sa5int,
Sa6int, and Sa7int. Further studies on integrase gene correlations were then conducted
with other characteristics such as amidase groups, phage types, morphogenesis subtypes,
etc. (Kahánková et al. 2010). Knowing the pattern between integrase genes and various
virulence factors, I decided to determine if integrase genes could be used as a method of
phage or bacterial characterization to predict resistance to bacteriophages.
11
Purpose of Experiment
The purpose of this research was to induce bacteriophages from their lysogenized
state and correlate their lytic ability to their integrase gene. There has yet to be a
consistent correlation between the lytic ability of a phage and any part of its genome.
Using the integrase genes described by Goerke et al. this experiment investigated if
integrase genes could be a viable option (2009). I also asked if the presence of specific
integrase genes on the host chromosome would be a strong indicator for bacteriophage
resistance of the host. The hypothesis was: if a bacterium harbored a prophage with a
particular integrase, other bacteriophages having the same integrase would not be able to
lyse it. Therefore, the bacterium would be labeled resistant to that phage.
12
CHAPTER THREE
Methods and Materials
Obtaining a Staphylococcus aureus library
Staphylococcus aureus isolates used for this study were obtained from nasal swabs of a
healthy undergraduate population at Baylor University. The swabs were cultured onto
Mannitol Salt Agar plates to test for fermentation. After the confirmation of a halophile
which fermented mannitol, the culture was Gram-Stained and tested for catalase and
coagulase activity. Gram stains confirmed Gram positive staphylococci. Cultures positive
for all the mentioned tests were confirmed to be S. aureus and were assayed for antibiotic
resistance through Kirby-Bauer Disc Diffusion. Cultures were then frozen on cryobeads
for storage.
Screening for Integrase Genes
One hundred and four isolates were screened for the 7 most common Siphoviridae
integrase genes by Polymerase Chain Reaction (PCR) as determined by Goerke et al.
(2009). Achromopeptidase Boiling Lysis (1 unit/μL) and the Qiagen DNA Spin Column
Kit™ were used to extract genomic DNA (Ezaki, T and S. Suzuki, 1982). Samples
selected for extraction displayed a diversity in antibiotic resistance patterns. A multiplex
PCR kit (Qiagen) was used to perform PCR for the integrase genes. PCR solutions
consisted of 10 ng of DNA, 12.5 μL of Qiagen multiplex master mix, 200 nM of each
primer (Table 3.1), and approximately 2-5 μL of nuclease free water to make a 25 μL
mixture. PCR products were analyzed by gel electrophoresis on a 2-3% agarose gel
13
stained with GelStar (Cambrex). The gel was subsequently photographed under UV light
and the integrase bands were identified for each isolate.
Integrase Gene
Sa1int [length, 569 base pairs]
Primers (forward and reverse)
Sa1-F (AAGCTAAGTTCGGGCACA)
Sa1-R (GTAATGTTTGGGAGCCAT)
Sa2int [length, 640 base pairs]
Sa2-F (TCAAGTAACCCGTCAACTC)
Sa2-R (ATGTCTAAATGTGTGCGTG)
Sa3int [length, 475 base pairs]
Sa3-F (TTATTGACTCTACAGGCTGA)
Sa3-R (TTATTGACTCTACAGGCTGA)
Sa4int [length, 320 base pairs]
Sa4-F (ATTGATATTAACGGAACTC)
Sa4-R (TAAACTTATATGCGTGTGT)
Sa5int [length, 375 base pairs]
Sa5-F (AAAGATGCCAAACTAGCTG)
Sa5-R (CTTGTGGTTTTGTTCTGG)
Sa6int [length, 167 base pairs]
Sa6-F (GCCATCAATTCAAGGATAG)
Sa6-R (TCTGCAGCTGAGGACAAT)
Sa7int [length, 214 base pairs]
Sa7-F (GTCCGGTAGCTAGAGGTC)
Sa7-R (GGCGTATGCTTGACTGTGT)
Table 3.1: The primers designed for the respective integrase groups (Goerke et al, 2007).
Phage Induction and Spot Assay
Frozen cultures from cryobeads or colonies from library plates were grown
overnight in Brain Heart Infusion (BHI) broth. Mitomycin C at a final concentration of 2
μg/μL was added to the overnight S. aureus cultures (Table 3.2). After 30 minutes of
incubation at 35 °C, pellets were obtained by centrifuging the cultures at 3000 × g for 10
minutes. Pellets were washed, re-suspended in fresh BHI broth, and incubated at 35 °C
for 4 hours. Cultures were then centrifuged again at 3000 × g for 10 minutes and the
supernatant was filtered through either a 0.22 or 0.45 μm membrane. The presence of a
lytic phage was tested by spotting the filtered solution onto a panel of 10 different S.
aureus isolates (Table 3.3). Each host strain of the panel was grown on a double-layer
agar, and 5 μL of the filtered phage solution was spotted directly onto the top agar. The
top agar was observed at 24 and 48 hours. Zones of clearing confirmed the presence of a
14
lytic bacteriophage within the induced solution. The panel, selected for their variation in
hosting different integrase genes, is shown in Table 3.3.
Isolate
1046
1705
1079
1130
1152
55
109
311
318
346
349
2040
2045
2057
2063
Integrases
2,3,5
3,6
3
2,3,5,6
1,3
3
2,5
1,3
3,5
3,6
6,7
3
2,7
3
3
Isolate
2069
2414
2418
2437
2447
2653
2671
2672
2695
2734
1240
1278
1307
1463
1470
Integrases
3,5
1,3
3
3
3
3
3,7
1,3
3
3
2,3
6
3,6
2,3
1,3
Isolate
240
371
374
405
398
2019
2035
2163
2195
2210
2263
2372
2419
2427
2431
Integrases
3
3
3
3,5
3
3
3
3,4
2
3
1,3
1,3
3
3
3,7
Isolate
2455
2458
2503
2669
2691
2768
2819
2860
2868
3017
3064
3097
3180
Integrases
3,5,6,7
2,3,5
1,2,3,5
3
3
3
3,5,6
3
3,7
1,3
3
1,2,3
3
Table 3.2: Fifty eight isolates used for phage induction and their corresponding genomic
integrase genes.
S. aureus Isolate
Integrases
ATCC
Not tested
2344
5
227
3,4,7
1026
2
349
6,7
2019
3
2553
1,2,3
1278
6
2072
1
311
1,3
Table 3.3: Filtered crude solutions from the induction procedure were all spotted against
each of these 10 S. aureus isolates to screen for the presence of a lytic phage in the
solution. The 10 isolates were chosen because of the diverse category of genomic
integrase genes.
15
Phage Purification, Plaque Assay and Determination of Host Range
Based on the results of the initial spot assay, 16 phage solutions were chosen to be
further investigated. Host strains were selected to propagate and purify these phages
based on the results of the panel screening and their susceptibility to specific phage
solutions. Two different strains (2553 and 311) were the hosts used to purify the phages
(Table 3.4). The final purified phages were labeled PhageBear1, PhageBear2,
PhageBear3 and Phagebear4.
Phage
Host Strain
PhageBear1
311
PhageBear2
2553
PhageBear3
2553
PhageBear4
2553
Table 3.4: Different strains were used to purify the phages. Staphylococcus aureus strain
311 was used for PhageBear1 and strain 2553 for PhageBear2, PhageBear3 and
PhageBear4.
Phages were then purified through 4-5 subsequent plaque assays using the doubleagar overlay method. A plaque was picked from the top agar layer and resuspended in 50
μL of phage buffer. From this solution, 5 μL was mixed with 500 μL of an overnight S.
aureus culture and incubated at room temperature for 10 minutes. Top Agar was added
and the solution was immediately plated on a Tryptic Soy Agar plate. Plaques that
appeared the following day were then picked and the procedure was repeated 4-5 times.
Quantification of the purified phage solution was measured by counting the
number of PFUs on the top agar after 24 or 48 hours of incubation at 37 °C. This number
16
was then divided by 5 μL, the amount inoculated into the culture, multiplied by the
dilution number, and converted to PFU/mL. Purified phages were all determined to have
a similar concentration of
PFU/mL. Spot tests for each purified phage were
performed on 64 different S. aureus isolates to determine the phage’s host range. Positive
tests for lysis on the spot test were then verified by plaque tests. Phage K, a well-known
bacteriophage, was used as a standard for confirming reproducibility between assays and
for comparison (O’Flaherty et al. 2004).
Phage DNA Extraction and Integrase Screen
To obtain a sufficient concentration of bacteriophages for DNA extraction, 6-8
web plates (a lawn of bacteria on top agar that is 95% lysed) were flooded with phage
buffer for 24 hours. The buffer was then aspirated and filtered through a 0.45 μm
membrane. Ten mL of the solution was incubated at 37 °C with nuclease for 30 minutes,
followed by incubation at room temperature for 1 hour. Phages were precipitated using a
phage precipitation solution containing 30% Polyethylene glycol and 3.3 M NaCl.
PEG/phage solutions were then incubated overnight at 4 °C and then separated by
centrifugation at 10,000 × g for 20 minutes. The supernatant was removed and the pellet
was resuspended in sterile water. DNA from the purified phages was then extracted using
the Promega DNA Extraction Kit, whereby a resin was added to uncoat the phage
particles and bind the DNA. Phage-resin purified solution was column purified using the
Promega columns provided, washed with 80% isopropynol, dried through centrifugation,
and eluted with warm 10:1 TE. The DNA was then quantified using a nanodrop
spectrophotometer and then analyzed for integrase gene types using PCR and gel
electrophoresis as described previously.
17
CHAPTER FOUR
Results
Prevalence of Integrase Genes in 104 S. aureus isolates
Each of the 104 S. aureus isolates screened for the presence of integrase genes
displayed at least one type of integrase based on the gel electrophoresis results (Figure
4.1, Table 4.1). The isolates chosen were a varied combination of isolates that were
resistant to methicillin (28 isolates), resistant only to penicillin (37 isolates), and
susceptible to all 12 antibiotics tested (39 isolates). The antibiotics tested on the Kirby
Bauer Disc Diffusion were Penicillin, Erythromycin, Clindamycin, Neomycin,
Doxycycline, Ciproflaxin, Trimethoprim Sulfa, Oxacillin, Rifampin, Gentamicin,
Amikacin and Nitrofurantoin.
With respect to the integrase genes screened for in the hosts, Sa3int was the most
prevalent integrase gene, found on 86.5% of all isolates, while Sa4int was the least
prevalent, and was only found on 1.9% of all isolates (Figure 4.2). When the results are
separated between MRSA and MSSA (Methicillin Susceptible S. aureus) isolates, there is
a slight difference in their prevalence (Figure 4.3). Conversely, Sa6int was not present in
any of the MRSA isolates (n=28) but was shown to be in 17.1% of the MSSA isolates
(n=76). The presence of integrase genes indicated the presence of prophages in all of the
isolates tested.
18
Well
1
2
3
4
5
6
7
8
9
10
11
12
13
Integrases
2,3
2,3
2,3,5
3,7
2,3
2,3
3
1,3
3,7
3
3
None
(cont.)
100 bp
Ladder
Sa2int
Sa1int
Sa5int
Sa3int
Sa7int
Figure 4.1: Gel electrophoresis on a 3% agarose gel to separate the different sized DNA
bands encoding various integrase genes (100bp ladder). As depicted, the most common
integrase gene was Sa3int (band length: 475bp).
Length
Integrase
167 bp
214 bp
320 bp
375 bp
475 bp
569 bp
640 bp
Sa6int
Sa7int
Sa4int
Sa5int
Sa3int
Sa1int
Sa2int
Table 4.1: The corresponding DNA band lengths for each S. aureus integrase type
(Goerke et al. 2007)
19
Prevalance of Integrase Gene Types among 104 MSSA and MRSA
isolates
100.0%
90.0%
80.0%
70.0%
60.0%
50.0%
40.0%
30.0%
20.0%
10.0%
0.0%
86.5%
31.7%
18.3%
18.3%
12.5%
14.4%
Sa5int
Sa6int
Sa7int
1.9%
Sa1int
Sa2int
Sa3int
Sa4int
Figure 4.2: All of the S. aureus isolates contained at least 1 prophage as detected by PCR
screening for integrase genes. There is a clear variation in prevalence of different
integrase types.
Percentage
Prevalence of Integrase Type Among 28 MRSA and 76 MSSA Isolates
100.0%
90.0%
80.0%
70.0%
60.0%
50.0%
40.0%
30.0%
20.0%
10.0%
0.0%
Total Percentages
Sa1int
18.3%
Sa2int
31.7%
Sa3int
86.5%
Sa4int
1.9%
Sa5int
18.3%
Sa6int
12.5%
Sa7int
14.4%
MRSA Percentages
17.9%
53.6%
92.9%
3.6%
14.3%
0.0%
17.9%
MSSA Percentages
18.4%
23.7%
84.2%
1.3%
19.7%
17.1%
13.2%
Figure 4.3: Differences between the types of integrase genes found in MRSA and MSSA
isolates. There was no difference between the percent of integrase genes Sa1int, Sa3int,
Sa4int, Sa5int and Sa7int present in both MRSA and MSSA isolates, however, MRSA
isolates had a 29.9% greater chance of harboring a phage with integrase Sa2int than
MSSA isolates. On the other hand, none of the MRSA isolates carried integrase Sa6int
even though it was found in 17.1% of the 76 MSSA isolates. Black bar: Total
percentages; Striped bar: MRSA percentages; Dotted bar: MSSA percentages.
20
Polylysogeny
Another point of interest was the prevalence of polylysogeny, whereby an isolate
harbors multiple types of phages. The study shows the least possible number of
prophages present, because the complete host genomes are not known and there may be
prophages not identified with the 7 integrase genes used. While 45 out of 104 isolates
possessed only one prophage, 37 isolates harbored 2, 16 isolates harbored 3 and 6 isolates
harbored 4 (Figure 4.4). The median for polylysogeny is two prophages per isolate
(Figure 4.4). The rate of polylysogeny was similar between MRSA and MSSA isolates
(Figure 4.5).
The Prevalence of Polylysogeny
Number of isolates
45
45
40
35
30
25
20
15
10
5
0
37
16
6
1
2
3
Prophages in
Chromosome (All)
4
Prophages in Chromosome
Figure 4.4: The prevalence of polylysogeny among 104 S. aureus isolates. The largest
number of different types of integrase genes found in a particular isolate was 4, which
was the case for 6 isolates.
21
Average Number of Lysogenized Bacteriophages
Number of Lysogenized Prophages
Average for All
2
Average for MSSA
1.83
1.77
Average for MRSA
2
1.5
1
0.5
0
Figure 4.5: The number of lysogenized bacteriophages within MRSA and MSSA were
similar. Out of 104 isolates the average number of lysogenized bacteriophages was 1.83
phages per isolate. When separated into MRSA and MSSA, the MSSA isolates average
1.77 prophages per isolate and the MRSA average 2.00 prophages per isolate. Solid bar:
Average for all; Striped bar: Average for MSSA; Dotted bar: Average for MRSA.
Phage Induction and Purification
Of the 104 S. aureus isolates screened for integrases, these 58 isolates were
chosen for phage induction. These 58 isolates were selected to include both variations in
number and type of integrases found in the host. Of the 58 isolates which underwent
Mitomycin C induction, 40 produced a filtrate that demonstrated the presence of lytic
phages, as detected by the spot test against 10 different S. aureus isolates (Table 4.2). The
16 filtrates with the largest infectivity against the 10 isolates were selected for
purification (Table 4.2). Initial plaques assays with the phage induction solution for each
of these 16 putative phage solutions produced plates with over 50 pinpoint sized plaques
(Figure 4.6). Attempts to produce a larger plaque through manipulation of time,
22
temperature, incubation period and media did not produce a change in plaque
morphologies (results not shown). Only 4 phages (PhageBear1, PhageBear2,
PhageBear3, and PhageBear4*) were successfully purified through 4-5 subsequent
rounds. The presence of plaques disappeared for the remaining 12 filtrates after the first
two purification rounds. Phages from the phage induction solutions that were unable to be
purified were: 1130, 2868, 2819, 3, 330, 55, 2045, 1470, 240, 2672,
2163, 2195**.
* Note in lab notebook that PhageBear1 = Φ2734; PhageBear2 = Φ346; PhageBear3 =
Φ349; PhageBear4 = Φ2069. Name changes were made in the paper for simplicity.
**  indicates phage solution. The number represents the host S. aureus strain that was
stressed into induction. After induction, the solution will not have a purified phage and so
 was added to distinguish the phage solution from the strain itself, as well as from the
purified phages (see note above)
Figure 4.6: Spot tests (left) of various phages on host strain 1127. (Right) PhageBear2
plaque assay; this picture shows the small, pinpoint plaques with diameters < 1mm.
23
Isolate
109
318
374
398
1079
1152
1240
1307
1463
1705
2019
2063
2210
2263
2372
2414
2418
2427
2437
2447
2455
2503
2669
2671
2691
2695
2768
2860
3017
3064
3180
311
349*
371
405
1046
1130
1278
2035
2057
2195
2419
2431
2458
2653
3097
55
240
346*
1470
2040
2045
2069*
2163
2672
2734*
2819
2868
Integrases Found
in phage solution
2,5
3,5
3
3
3
1,3
2,3
3,6
2,3
3,6
3
3
3
1,3
1,3
1,3
3
3
3
3
3,5,6,7
1,2,3,5
3
3,7
3
3
3
3
1,3
3
3
1,3
6,7
3
3,5
2,3,5
2,3,5,6
6
3
3
2
3
3,7
2,3,5
3
1,2,3
3
3
3,6
1,3
3
2,7
3,5
3,4
1,3
3
3,5,6
3,7
ATCC
2344
227
1026
349
2019
2553
1278
2072
311
3
1
3
2
3
2
2
1
2
3
1
3
3
1
1
2
2
2
2
3
3
3
1
2
2
2
2
1
2
2
2
3
2
1
2
2
1
2
2
2
3
3
3
3
2
3
3
2
3
2
3
2
2
3
2
2
3
3
3
3
2
2
1
3
3
3
3
3
3
3
3
2
1
3
2
3
3
2
2
3
3
2
3
2
3
3
2
2
3
3
2
3
3
1
3
3
1
3
2
3
3
3
2
2
2
1
1
2
2
1
2
3
2
1
2
2
3
3
1
2
3
2
2
3
3
2
1
2
1
3
2
2
1
2
3
2
2
2
3
2
1
2
2
3
3
3
3
2
2
2
3
3
2
3
1
2
2
1
3
3
2
2
2
1
2
2
2
2
2
1
1
Table 4.2: The 58 induced solutions were spotted onto a panel of 10 isolates. Results of
the spot tests were described as follows: 3 (turbid), 2 (semi-turbid), 1 (clear spot and
strong lysis), - (no lysis). Number of strains lysed: Green (0-2), Blue (3-5), Yellow (610). The phages in bold red indicate that they were chosen to be purified. Phages in bold
red and with asterisks indicate that they were the final four phages that completed
purification and were tested for their host range.
24
Host Range of Induced Phages
Following purification, the 4 purified phages (PhageBear1, PhageBear2,
PhageBear3, and PhageBear4) were spot assayed for the number of S. aureus they could
lyse on a diverse set of 73 S. aureus isolates. Plaque assays confirmed lysis. PhageBear3
and PhageBear4 produced the widest host range of the four phages as they lysed 10.96%
of the isolates (Figure 4.6). However, PhageBear3 lysed more MRSA strains than
PhageBear4. PhageBear1 had the smallest host range as it only lysed 5.48% of all of its
isolates. The standard for this experiment was phage K, which lysed 83.56% of the 73
Percentage of
Strains Lysed
isolates.
Phage Host Ranges
100.00%
90.00%
80.00%
70.00%
60.00%
50.00%
40.00%
30.00%
20.00%
10.00%
0.00%
PhageBe
PhageBe
PhageBe
PhageBe
ar1
ar2
ar3
ar4
Total (n=73)
83.56%
5.48%
6.85%
10.96%
10.96%
MRSA (n=23) 86.96%
4.35%
13.04%
13.04%
8.70%
MSSA (n=50)
82.00%
6.00%
4.00%
10.00%
12.00%
Figure 4.6: The host range, or the range of infectivity, of the 4 purified phages against 73
S. aureus isolates. A spot test was performed for each phage, and a plaque assay was used
to confirm lysis. Phage K, a well-known lytic phage, was used as a standard to compare
the host ranges of the induced phages. Statistical analysis shows no difference between
MRSA and MSSA strains vs. phage infectivity. Pearson p-values: 0.5956, 0.7732,
0.1553, 0.6990, 0.6746 (from left to right, as depicted by the graph). Solid bar: percent
lysed total; Striped bar: percent MRSA lysed; Dotted bar: Percent MSSA Lysed.
K
25
CHAPTER FIVE
Conclusion and Discussion
Phage Induction and Purification
Of the 58 induced phage solutions, 40 produced lytic phage via the initial spot
assays. The sixteen solutions that exhibited either high lytic capability or were induced
from strains with a variety of integrase genes were analyzed using plaque assays. Four of
the 16 solutions produced plaques at the end of plaque purification. The absence of
plaques from the remaining 12 solutions may be attributed to low phage concentrations
after phage plate to buffer transfer. Another possibility is that the host bacterium is killed
before the incoming phage is able to propagate itself, therefore only allowing the phages
to infect cells through the spot assay but not through the plaque assay (Allison and
Klaenhammer, 1998). Abortive infections have also been known to reduce plaque sizes.
This may be the cause for the small plaque morphologies formed from the PhageBear
phages. The plaques from these phages had diameters no larger than 1 mm (Figure 5.1)
and were two to three times smaller in size than those from Phage K.
26
Figure 5.1: Plaque assay of PhageBear3 depicting the small plaque sizes. Most plaques
displayed a diameter no larger than 1 mm.
Phage Host Range and Resistance
With respect to the host range, the four induced and purified phages displayed
lytic abilities much less than that of the standard, Phage K. Moreover, Phage K lysed
83.56% of all isolates, which is a higher percentage than previous reports (O’Flaherty et
al. 2005). The difference in percentages may be attributed to different samples, but
further investigation is necessary. As for the significantly smaller host ranges of the
induced phages (PhageBear1, PhageBear2, PhageBear3, PhageBear4), the low lytic
activity may be due to the phages originally being lysogenized phages that were induced
for lytic activity.
A main aspect of this experiment was to determine if the presence of particular
integrase genes within the host chromosome could predict bacteriophage resistance for
specific strains. The results indicate that the type of integrase genes does not provide a
27
correlation for resistance. For example, PCR confirmed that all 4 of the PhageBear
phages had integrase Sa6int but they lyse both S. aureus strains that had Sa6int in their
chromosome and those which did not.
Statistical analysis using Pearson p-values also tested whether a correlation exists
between phage resistance and the presence of integrase Sa6int in bacteria. When
comparing the lytic ability of the five phages to the susceptibility of hosts that harbored
Sa6int, 4 of the 5 contingency analyses showed Pearson p-values > 0.05 (Figure 5.2). The
p-value for PhageBear1 with 0.0446 indicates that there may have been a correlation
between PhageBear1 and the susceptibility of bacteria with integrase Sa6int. However,
when examining the sample sizes (only 4 isolates were susceptible to PhageBear1
compared to 69), it is difficult to conclude that such a correlation exists. More samples
will be needed to validate such a result.
Statistical analysis for strains harboring the other integrase genes (Sa1int, Sa2int,
Sa3int, Sa4int, Sa5int, Sa7int) displayed similar results. Conclusions from these results
show that the type of integrase gene, Sa1int-Sa7int, is not a valid predictor for phage
resistance.
The number of integrase genes found in the host chromosome also cannot predict
phage resistance. In addition, the method of detecting integrase genes does not ensure
that all integrase genes in the genome are detected. Figure 5.3 illustrates the phage
susceptibility similarities among strains that harbored at least 1 integrase gene, 2
integrase genes, and 3 or more integrase genes.
The presence of integrase Sa6int in all four phages was unexpected since
integrase Sa3int was the most abundant integrase gene in host genomes. When a PCR
28
was performed on the DNA of the S. aureus isolates from which the phages were
induced, integrase gene Sa6int was present only in two of the four isolates. Isolates that
produced PhageBear1 and PhageBear4 did not have integrase Sa6int. This raises the
question on where the phages came from and why they all have the same integrase gene.
We can eliminate possibility of contamination because the varying host ranges of the
phages prove that they are, in fact, different phages. Purifying four phages of the same
integrase type may indicate a higher prevalence of lytic activity with integrase Sa6int. As
for the inconsistency between the Sa6int integrase genes of the phages and the absence of
the Sa6int integrase gene of their lysogen, I attribute this to difficulties the lack of PCR
sensitivity, which requires further research.
Strains
Strains
Pearson
Susceptible
Resistant
p-val.
Sa6int
Sa6int
Sa6int
Sa6int
Present
Absent
Present
Absent
Phage K
10
51
1
11
0.4756
PhageBear1
2
2
9
60
0.0446
PhageBear2
0
5
11
57
0.3291
PhageBear3
2
6
9
56
0.4053
PhageBear4
2
6
9
56
0.4053
Figure 5.2: Results of contingency analyses of induced phages vs. bacteria harboring
integrase Sa6int. Though PhageBear3 and PhageBear4 have quantitatively similar lytic
infectivity results, the strains they lysed were different, thus marking them different
phages.
29
Effects of Polylysogeny on Phage Susceptibility
100%
90%
80%
70%
60%
Percent of 50%
strains lysed 40%
30%
20%
10%
0%
Phage K
PhageBear1
PhageBear2
PhageBear3
PhageBear4
1 Harbored (n = 26)
77%
8%
4%
8%
12%
2 Harbored (n = 33)
88%
3%
9%
15%
12%
3+ Harbored (n = 14)
86%
7%
7%
7%
7%
Figure 5.3: The host strains were divided into three categories: those that harbored only 1
integrase gene (n=26), those with 2 (n=33), and those with 3 or more (n=14). The strains
in the respective categories were compared to their susceptibility to each of the purified
bacteriophages. Phage K was the standard for the experiment. Solid bar: 1 phage
harbored; Striped bar: 2 phages harbored; Dotted bar: 3+ phages harbored.
Final Remarks
Given that integrase genes are not correlated to lytic ability, the need still exists to
describe the behavior of these phages against bacteria through a type of genetic
identification. If phages can be classified by their infectivity based on their genetic
material, the search for highly lytic phages can be simplified and reduced to merely
screening for a particular gene.
Furthermore, the link between bacterial genomes and phage resistance patterns
must be addressed. Previous research on phage-bacteria interactions has postulated
several reasons and mechanisms for phage resistance. Studies have connected phage
resistance to a multitude of variables including: the presence of clustered regularly
30
interspaced short palindromic repeats (CRISPR) genes (Barrangou et al. 2007), the
inability of phages to bind to the receptors on bacterial cell walls (Ohshima et al. 1988;
Wilkinson and Holmes, 1979), and superinfection exclusion (Hyman and Abedon, 2010).
This research concluded that integrase genes may not have a function in phage resistance.
By further investigating the different methods bacteria can become resistant to phages,
these phage-bacteria interactions can be better understood and lead to the production of
highly lytic phages.
Future research could consist of co-inoculating phages with several different
compounds to observe changes in lytic activity. Ambient factors, such as pH and
temperature, and their relation to phage activity are also potential future areas for
research in this field. Comparing these phages on a genomic level is also another avenue
for future research.
Moreover, future research can investigate the lytic ability of bacteriophages on
strains from various sources. The difference in our reported host range for Phage K and
other reports indicate that the source of the host strains may play a role in susceptibility.
Sources can include environmental, nosocomial, and anatomical samples.
Understanding the characteristics of bacteriophages will be important in the
development of an effective treatment against antibiotic resistant bacteria. With the
emergence of vancomycin resistant S. aureus and the increasing prevalence of MRSA,
bacteriophage therapy is becoming a more appealing option. The scientific community
will need to acquire more information on bacteriophages in order for them to be utilized
to stop infections and improve human health.
31
APPENDICES
32
APPENDIX 1
LIST OF RECIPES
Blood Heart Infusion (BHI) Broth
500 mL D.I. Water
18.5 grams BHI base
7H9 (Middlebrook broth)
2.35 grams 7H9 Middlebrook broth base
500 uL D.I. Water
Tryptic Soy Agar (TSA)
20 grams TSA Agar
500 mL D.I. Water
Luria Agar
7.75 grams Luria Base
7.5 grams Agar
10 grams NaCl
1X Top Agar
100 mL 2X Top Agar
100 mL 7H9
2 mL Calcium Carbonate
2X Top Agar
2.35 grams 7H9 Base or 18.5 grams BHI base
4 grams Agar
500 mL D.I. Water
50X TAE Stock Solution
242 grams Tris Base
57.1 mL Acetic Acid
100 mL 0.5 EDTA
1X TAE
20 mL 50X TAE
980 mL D.I. Water
33
2% Agarose Gel
0.6 grams Agarose
30 mL 1X TAE Buffer
1 uL GelStar Stain
Polymerase Chain Reaction Mixture
12.5 uL of 2X Qiagen PCR Mix
200 nM of each Primer
4.5 uL of RNase free water
10 ng water
Polymerase Chain Reaction Thermocycler Settings
95°C for 15 minutes
35 cycles of amplification (95°C for 30 seconds, 55°C for 60 seconds, 72°C for
45 seconds)
72°C for 10 minutes
Hold at 4°C
Ran at 85 volts for 80 minutes
Achromopeptidase (1 Unit/uL)
22.1 mg ACP (4527 Units/mg)
100 mL D.I. Water
Mitomycin C (1 mg/mL)
2 mg Mitomycin C
2 mL D.I. Water
100 mM CaCl2
1.11 grams of Anhydrous CaCl2
100 mL D.I. Water
1 M MgSO4
120.36 grams MgSO4
1 L D.I. Water
Nuclease Mix
0.088 grams NaCl
4.25 mL D.I. Water
500 uL DNase I (5 mg/mL)
250 uL RNase A (10 mg/mL)
5 mL Glycerol
34
Phage Buffer
10 mL 1 M Tris stock (pH 7.5)
10 mL 1M MgSO4
4 grams NaCl
970 mL D.I. Water
10 mL 100 mM
Phage Precipitation Solution
100 mL D.I. Water
30 grams Polyethylene glycol 8000 (PEG 8000)
19.3 grams NaCl
35
APPENDIX II
Protocols
Achromopeptidase Boiling Lysis
1.
2.
3.
4.
5.
6.
7.
8.
Grow overnight culture in BHI broth.
Transfer 1.5 mL of overnight culture to a microcentrifuge tube.
Centrifuge of culture at 3000 × g for 10 minutes. Discard supernatant.
Repeat steps 2 and 3.
Add 200 μL of Achromopeptidase (995 U/mL) to resuspend cell pellet.
Incubate in 37°C for 10 minutes.
Transfer microcentrifuge tubes to a boiling water bath for 5 minutes.
Wait for the tubes to cool and transfer the supernatant (DNA) into a new
microcentrifuge container for storage.
Prophage Induction – Mitomycin C (1 μg/μL)
1. Grow cultures in 5 mL BHI Broth to an Optic Density at 600 nanometers of 0.1
(~2 hours)
2. Add 10 μL of Mitomycin C (1 μg/μL) for a final concentration of 2 μg/μL
3. Incubate for 30 minutes
4. Centrifuge cultures for 10 minutes at 3000 × g to retrieve cell pellet
5. Resuspend pellets in fresh BHI broth
6. Centrifuge cultures for 10 minutes at 3000 × g to retrieve cell pellet
7. Resuspend pellets in new 3 mL BHI broth
8. Incubate for 4 hours at 35°C
9. Centrifuge cultures for 10 minutes at 3000 × g to retrieve cell pellet
10. Filter supernatant through a 0.22 μm or 0.45 μm membrane and store potential
phage solution.
Phage Induction – Screening Phages via Spot assay
1. Prepare a panel, 8-10 different isolates, of host strains to test the potential phage
solution by growing cultures in 5 mL BHI Broth overnight.
2. Following overnight growth, aliquot 500 μL of each isolate into a separate tube.
3. Add 4.5 mL of 1X Top Agar to the culture
4. Pour contents onto a TSA plate
5. Let the Top Agar cool by incubating it at room temperature for 10 minutes
6. Perform a spot test with your potential phage solution by spotting 5 μL directly
onto the Top Agar
36
7. Incubate overnight at 35°C
Phage Purification (From Potential Phage Solution)
1. Add 5 μL of potential phage solution to 500 μL of S. aureus culture
* Note: The isolate chosen should be one that was confirmed to have been lysed
by the solution in the spot assay
2. Incubate at room temperature for 25 minutes
3. Add 4.5 mL of 1X Top Agar (w/ 1% CaCl)
4. Pour contents onto a TSA plate
5. Let the Top Agar cool by incubating it at room temperature for 10 minutes
6. Incubate plate at 35 °C overnight
7. Check for plaques the following day (24-48 hrs after incubation)
Phage Purification (Subsequent Purification Steps)
1. Grow desired number of host strains to test the lytic phage against in 5 mL BHI
Broth overnight.
2. Following overnight growth, aliquot 500 μL of each isolate into a separate tube.
3. Add 4.5 mL of 1X Top Agar (w/ 1% CaCl) to the culture
4. Pour contents onto a TSA plate
5. Let the Top Agar cool by incubating it at room temperature for 10 minutes
6. Perform a spot test with your potential phage solution by spotting 5 μL directly
onto the Top Agar
7. Incubate overnight at 35°C
Host Range Assay
1.
2.
3.
4.
5.
6.
7.
8.
Add 500 μL of overnight Staph into a small test tube
Inoculate the Staph with 10 μL of phage
Incubate at room temperature for 25 minutes
Add 4.5 mL of 1X Top Agar (w/ 1% CaCl) to mixture and pipette contents onto
TSA plate
Swirl the plate gently to ensure the Top Agar forms a thin layer throughout the
plate.
Let the Top Agar sit on the plate for 5-10 minutes so that it can solidify
Flip the plate over and incubate overnight at 35°C.
Check for plaques the following day (24-48 hrs after incubation)
37
Phage DNA Extraction – Promega Kit
1.
2.
3.
4.
Allow 4.5 mL of Phage Buffer to sit on top of web plates.
Place plates in refrigerator overnight.
Add another 4 mL of Phage Buffer and incubate in room temperature for 1 hour
Aspirate the phage in phage buffer solution from the plate using a syringe and
aliquot contents into a filter sterilizing vacuum. Filter sterilize the contents.
5. Aliquot 10 mL into a separate tube and add 40 μL of Nuclease Mix
6. Incubate at 35°C for 30 minutes
7. Incubate at room temperature for 1 hour
8. Add 4 mL of Phage Precipitation Solution
9. Incubate overnight in 4°C
10. Centrifuge tube at 10,000 × g for 20 minutes
11. Discard supernatant
12. Add 0.5 mL of sterile D.I. water and re-suspend pellet
13. Incubate at room temperature for 5-10 minutes
14. Add 2 mL of pre-warmed (37°C) DNA Clean Up Resin and gently swirl the tube
15. Apply 1.25 mL of water-resin-phage-genomic-DNA solution to a DNA Extraction
kit column using a pipette
16. Use the syringe plunger to push this solution through the column
17. To wash salts and proteins off of the column
a. Remove the column
b. Remove the plunger
c. Re-attach syringe
d. Add 2 mL of 80% isopropanol and push the solution through the column
with the plunger
18. To dry the column
a. Remove the column
b. Centrifuge the column for 5 minutes at maximum speed to remove the
isopropanol
c. Transfer the column to a clear microcentrifuge tube
d. Centrifuge at 1 minute at full speed to remove the residual alcohol
19. Elute the phage genomic DNA from each column:
a. Transfer the column to a sterile microcentrifuge tube
b. Apply 50 μL of pre-warmed, 80°C, 10:1 TE buffer to the resin in the
column and let it sit for 30-60 seconds to dissolve the DNA
c. Centrifuge for 1 minute at high speed to elute the DNA
20. Store DNA in a 4°C refrigerator.
38
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