King Saud University
Faculty of Science
Department of Botany and Microbiology
The Role of Staphages in The Treatment of
Methecillin Resistant Staphylococcus aureus
Infection
By:
Eman Kamel Al-Digs
Submitted in partial fulfillment of the requirements for the Doctor of Philosophy Degree
in the Department of Botany and Microbiology at the Faculty of Science, King Saud
University
Supervised By:
Dr. Nagwa Mohamed Amin Aref
1425-2004
‫الحمد هلل رب العالمين‬
To My Family…
To My Friends…
Thank you for all what you did for me, it means so much
‫أتقدم بالشكر لمدينة الملك عبد العزيز للعلوم والتقنية‬
‫لدعمها لهذا البحث بمنحة رقم‬
‫أ ط – ‪77 - 8‬‬
ACKNOWLEDGMENTS
The author wishes to express her sincere appreciation to many people who made this endeavor
possible.
Acknowledgment is due to King Saud University and King Abduaziz University for support of
this research.
Very special thanks to Dr. Nagwa Aref , Prof. of Virology and my major prof. at
the Department of Botany and Microbiology , Faculty of Science , King Saud
University for her supervision ,advise , and guidance for this investigation. I would
like to thank Dr. M. Hussein for supervision in this work.
I would like also to acknowledge Dr. A. Al- Farhan for his understanding, guidance and
support.
Very sincere thanks go to Dr. M. Al-Ahdal, Dr. S. Althawadi, and G. Kessie;
King Faisal Speciallist Hospital and Research Center. Dr. B. Taj, National Guard
Hospital; for their helpful discussion, encouragement and providing all necessary
facilities and materials.
My appreciation goes to all staff members of the department of Botany and
Microbiology, Faculty of Science, King Saud University; for their great help and
guidance, especially Dr. A. Abu-Taleb.
My appreciation is also extended to all hospitals and polyclinics who
cooperated in this study, especially Dr. A. Saraweq; King Fahd General Hospital /
Jeddah.
A special pray for my father who supported me in every step in this work
until it is here I shall be for ever grateful to him.
To all those who have assisted in this work “THANK YOU VERY
MUCH“.
Introduction
ii
Introduction
The Staphylococci are ubiquitous colonizers of human, animal
skin
and
mucous
membranes
causing
a
variety
of
syndromes.
Staphylococcus aureus is considered the most pathogenic organism. The
wide spread of antibiotic resistance among strains of S. aureus is a major
concern in the treatment of Staphylococcus infections. It is well known that
the organism acquires resistance soon after the introduction of new
antibiotics (Lyon and Skurray, 1987). As the incidence of antimicrobial
resistance rises, so do costs associated with its consequences. According to
estimates, the cost of resistance ranges from $75 million to $ 7.5 billion per
year. One study by the Centers for Disease Control and Prevention (CDC)
concluded that for both nosocomial and community-acquired infections,
those involving drug-resistant strains were at least twice as likely to be
associated with morbidity, hospitalization, and increased length of hospital
stay as those with drug-susceptible strains. Although it can only be roughly
quantities, antimicrobial resistance clearly is an important health problem
and an economic burden to society.
Methecillin was developed in 1960 for the treatment of such multidrug resistant S. aureus. However in the same year, Jevons (1960)
discovered Methecillin Resistant S. aureus (MRSA), which by 1970s
iii
became spread all over the world; but remained uncommon until 1986 when
a marked increase occurred in the number of isolates referred to the
Communicable Disease Group Institute of Environmental Science and
Research at Porirua (Rajan et al., 2002; and Ito et al., 2003).
In Jeddah during 1993 – 1994 Al -Aamodi (1997) found 40.4 % of the S.
aureus isolates was MRSA. In the other hand Al -Shammary (1997), in
Riyadh revealed 7.2 % MRSA infections from five different hospitals. While
the most frequently isolated pathogen from chronic otitis media swabs was
36% S. aureus in the study of Al- farraj (2002) in Riyadh.
Bacteria resistant to most or all available antibiotics are causing
increasingly serious problems, raising widespread fears of returning to a preantibiotic era of untreatable infections and epidemics.
There was a renewed interest in the possibilities of bacteriophage
therapy, where a specific kind of viruses that attack only bacteria used to kill
the pathogenic microorganisms (Levin and Bull, 1996; Lederberg, 1996;
Radetsky, 1996; Barrow and Soothhill, 1997).
Phages are efficacious antibacterial agents due to the lethality and
specifity of phages for particular bacteria. The ability to replicate within
infected animal hosts, and the boi-safety of phages made them efficacious
antibacterial agents. It is likely appeared that phage therapy would regain a
iv
role in both medical and veterinary treatment of infectious diseases
(Duckworth et al., 2002). The concept of bacteriophage therapy is simple;
bacteriophages are administrated to the infected animals or humans in an
effort to reduce populations of pathogenic bacteria through the natural
ability of phage to target and destroy bacterial cells. Although
bacteriophages had not been extensively used as antimicrobial agents since
the emergence of antibiotics, the rise of multiple antibiotic resistant strains
had a renewed interest in phage therapy (Platt et al .,2003). In making the
choice to explore the possibilities of phage therapy, it should also considered
their many potential advantages: 1. They are both self-replicating and selflimiting, since they will multiply only as long as sensitive bacteria are
present and then are gradually eliminated from the individual and the
environment. 2. They can be targeted far more specifically than can most
antibiotics to the specific problem bacteria, causing much less damage to the
normal microbial balance in the body. The bacterial imbalance or
"dysbiosis" caused by treatment with many antibiotics can lead to serious
secondary infections involving relatively resistant bacteria, often extending
hospitalization time, expense and mortality. 3. Phages can possibly be
targeted to receptors on the bacterial surface which are involved in
pathogenesis, so that any resistant mutants are attenuated in virulence. 4.
Few side effects have been reported for phage therapy. 5. Phage therapy
v
would be particularly useful for people with allergies to antibiotics. 6.
Appropriately selected phages can easily be used prophylactically to help
prevent bacterial disease in people or animals at times of exposure, or to
v
sanitize hospitals and help protect against hospital-acquired (nosocomial)
infections. 7. Especially for external applications, phages can be prepared
fairly inexpensively and locally, facilitating their potential applications to
underserved populations. 8. Phage can be used either independently or in
conjunction with other antibiotics to help reduce the development of
bacterial resistance. 9. For localized uses, phage has the special advantage
that they continue multiplying and penetrating deeper as long as the
infection is present, rather than decreasing rapidly in concentration below
the surface like antibiotics.
This work was preformed in tow categories, First: identification of S.
aureus samples which were collected from different microbiology labs in
Riyadh hospitals to determine their susceptibility to antibiotics, then
identifying the isolates with phage typing and molecular typing by pulsed
field gel electrophoresis. Second: Characterization of new phages that were
isolated by induction with ultra violet radiation to study their plaque
morphology, spectrum of infectivity, use of electron microscopy to examine
the morphology of isolated phages and identification the nature of the
phages nucleic acid by enzyme digestion and pulsed field gel
electrophoresis.
As such, the objectives of the present study were in to:
I. Monitoring the susceptibility of the collected studied isolates of S. aureus
vi
to different antibiotics.
II. Identifying the investigated S. aureus isolates with standard International
Phages Set (IPS), (Phage Typing).
III. Molecular typing for S. aureus for the more identification of the nontypable isolates after typing with the IPS.
IV. Induction of new phages from Saudi local S. aureus isolates, with ultra
violet radiation.
V. Isolation of temperate and virulent phages after induction according to
their effect on different S. aureus strains.
VI. Analyze the spectrum of the isolated phages infectivity for S. aureus
isolates.
VII. Provide morphological and molecular characteristics of the new
induced S. aureus phages.
VIII. Provide base line data for application of phage therapy for the first
time in Saudi Arabia and Middle East, to evaluate the efficacy of phage
therapy in medical cases of methecillin resistant S. aureus skin infection in
patients of wounds (surgical and accidental), diabetic foot infections, burn
and abscess cases since MRSA is one of the most resistant organisms for
antibiotics.
Abstract
Abstract
Over a period of one year (from February 2000 to may 2001)
173 Staphylococcus aureus isolates collected from microbiology
lab hospitals in Riyadh and Jeddah. Distribution of Methecillin
Resistant Staphylococcus aureus (MRSA) isolates among clinical
departments and isolations from different specimens, showed that
the wound infections resemble the highest percentage of the
clinical sites (26%). The all over percentage of MRSA topical
infections (70%) was higher than MSSA infections. All isolates
were sensitive to vancomycin which is the drug of choice for
MRSA infections. Mupirocin showed low degree of resistance,
which was 6%. Susceptibility to other commonly used antibiotics
was variable. Resistance oxacillin was the highest 34%. Some
degrees of resistance to sulphomethoxazole / trimethoprim and
fusidic acid (34%) were noticed. Terms like boarder line resistant,
low-level mutation, and boarder- line susceptible had also been
obtained in our studied isolates of S. aureus for methecillin using
Minimum Inhibitory Concentration test; which was observed in the
percentage of MRSA isolates in our data that had MICs of
256µg/m1 (30.83%). Studying the distribution of phage type
patterns among Staphylococcus patients isolates in Saudi cohort; it
was found that only 30.56% were typable with different degree,
and 69.7% were non typable. Differentiation increased in the
phage typing, and genome typing yielding 20 and 23 different
S.aureus types, respectively. A 61.71% of the MRSA isolates were
genetically typed by PFGE resulting one type of MRSA with one
genetic difference. Genome typing by PFGE was a powerful tool
not only for strain identification but also for the resolution of
clonal relationships of S. aureus strains. Observation on UV
induction of a S.aureus strains showed that the number of infective
centers increased by more than 100%. As seen in our electron
micrographs, our studied phages designated from ph1 to ph10; five
of these phages ph2, ph4, ph5, ph7, and ph10 were morphotype A,
present in Myoviredae group, their DNAs were in the range
43.6kb-48.5kb that is very close to this size range with minor
differences in phage strain. Ph9 and ph10; had more than one band
in their pattern. A mixture of ten phages differed in their virulence
to MRSA isolates were used with all patient isolates (30) that were
lysed completely by spot-test. Phage therapy for 30 patients of
MRSA in different skin infection cases, showed that phage therapy
were highly effective. Using a mixture of 10 phages daily (7-10
days), treatment gave us a close success rate of 90%.
Table of Contents
Page #
I.
II.
Introduction.
Review of Literature
Antibiotic Resistance
-The over prescribing of antibiotics
7
- Emergence of multiresistant S. aureus
9
- The importance of MRSA
12
 Phage typing
15
- Requirements of a typing method
15
- Phage typing of S. aureus.
17
- S. aureus epidemic types
19
- Staphylococcus carrier state.
21
- Selection of typing phages.
22
 Molecular typing by pulsed field gel
electrophoresis.
 Induction in lysogenic S. aureus.
- Bacteriophage plaque morphology.
23
27
30
 Electron microscopy of staphages
32
-Classification and frequency in staphages.
 Characterization of phage nucleic acid.
35
 Phage therapy.
- Historical aspects of phage therapy.
38
- Specific problems in early phage therapy work. 40
- Properties of phages subjected to therapy.
- Bacteriophages as antibacterial agents.
42
- Applying phage therapy in humans world
wide.
III.
43
1. Polish academy of Sciences, Worslaw.
43
2. Soviet Research on phage therapy.
50
3. British work.
54
4. French company L`Oreal.
56
5. U.S.A work.
56
MATERIAL and METHEODS :
1. Sample collection and Bacterial Isolation.
62
a. Identification of S. aureus Sample.
62
1..2 Pure bacterial cultures for propagation.
1..3 Glycerol cultures for preservation.
63
b. Antibiotic susceptibility tests:
2.1.
Disc diffusion method.
2.2.
Minimum Inhibitory Concentrations (MICs)
2.3.
Detection of MRSA homogenicity /
Heterogenicity.
64
64
3. Typing the Staphylococcus aureus isolates:
3.1.
Phage typing for the 69 MRSA isolates
3.2.
Molecular typing by Pulsed Field Gel
3.3.
63
65
65
electrophoresis.
66
3.3.1. Selection of bacterial isolates.
67
3.3.2. Preparation of agarose embedded
3.3.3.
bacteria.
3.3.4. Lysis step.
68
3.3.5. Proteolysis step.
68
3.3.6. Restriction digestion step.
68
3.3.7. Preparation of 1% agarose gel.
69
3.3.8. Casting the gel.
69
3.3.9. Electrophoresis conditions.
69
4. Isolation of new Staphylococcus phages.
4.1.
67
Detection of lysogeny by cross – culture.
70
70
4.2.
Isolation of phages from sewage samples.
70
4.3.
Ultra-Violet induction of lysogenic S.aureus.
71
4.3.1. Induction on plate.
71
4.3.2. Induction on broth.
71
5. Phage propagation.
5.1.
72
Selection of sensitive S. aureus strain to
5.2.
studied phages for propagation.
72
5.3.
Propagation on cultures of agar plates.
72
5.4.
Propagation on broth cultures.
73
5.5.
Routine Test Dilution (RTD).
73
5.6.
Phage preservation.
74
5.7.
Spectrum of isolated phage infectivity.
74
6. S. aureus phages characterization.
74
6.1. Precipitation of the phage by Poly Ethylene Glycol. 74
6.2. Electron Microscopy examination.
75
6.3. Characterization of phage Nucleic acid by Agarose 75
Gel Electrophoresis
8.1.
Isolation of phage DNA for PFGE.
75
8.2.
Electrophoresis conditions.
75
9. Phage Therapy.
9.1. Evaluation of phage efficacy for phage
therapy In vitro.
76
9.1.1. Preliminary phage concentration
76
9.1.2. Phage stock.
77
9.1.3 Preparation of phage dressing.
77
9.2. In vivo phage dressing therapy.
IV.
9.2.1 Skin infection cases.
77
9.2.2. Clinical Application of phage
78
dressing therapy.
78
RESULTS:
a. Staphylococcus isolates.
1.1.
2.
81
S. aureus identification.
81
Antibiotic susceptibility test.
81
MRSA screening.
81
1.2.
Disc diffusion method.
89
1.3.
Minimum inhibitory concentrations (MICs).
89
1.4.
MRSA homogenicity / hterogenicity.
99
3.1. Phage typing.
99
3.2. PFGE.
102
4.2. Phage isolates from sewage samples.
106
4.3.1. Induction on plate.
109
4.3.2. Induction on broth.
109
5.6. Spectrum of isolated phages infectivity.
116
7. Electron Microscopy.
118
8. Phages Nucleic Acid.
130
9.1 Phage therapy in vitro.
133
9.2. Phage therapy in vivo
136
I. Staphage therapy for MRSA in diabetic foot cases.
136
II. Staphage therapy for MRSA in post-operative infections.
138
III. Staphage therapy for MRSA in accedintal septic wound
Infections.
146
IV. Staphage therapy for MRSA in abscess cases.
150
V. Staphage therapy for MRSA in burn cases.
150
IV. DISCUSSION.
155
V.
Appendix I .
Appendix II .
VI.
Refrences.
Arabic summary.
List of Figures
List of Tables
Table#
2
Page#
Numbers of S. aureus isolates from each clinically
infected site and thedistribution of 173 S. aureus
isolates on different clinical site.
71
3
Antibiogram of S. aureus to all testedantibiotics.
76
4
S. aureus Methecillin MICs .
78
5
A comparison between criteria of local MRSA solated
Antibiogram typing phage typing and molecular typing.
87
6
Morphological Charecteristics of staphages.
101
7
Chracterization of Staphages induced from local Isolates
DNA size, Morphology, plaques, Range of activity,
bacterial isolate site, typing and induction.
8
104
Staphage therapy in MRSA diabitic foot infection cases
Staphage therapy for MRSA in Post operative infection cases. 115
9
Staphage therapy for MRSA in accidental septic Wound
infection cases.
118
10 Staphage therapy for MRSA in abscess cases.
120
11
121
Staphage therapy for MRSA in burn cases.