Download bacteriophage

BACTERIOPHAGE
Advanced Microbiology
Prof. Rola Darweesh
Done by:
Wea’am Quraan
Sara Alsouri
Marah Ibrahim
Sohib Alsaleh
Marah Madain
Introduction
In the golden age of the discovery of antibiotics, innumerable lives were saved. These highly potent “miracle”
drugs are no longer as effective as they were a half a century ago [1]. We are now entering an era in which
bacterial infections, such as septicemia and ventilator-associated pneumonia, might no longer be successfully
treated with antibiotics [1]. A few years ago, New Yorker magazine aptly satirized American attitudes toward
antibiotics with a cartoon of a doctor's office sign: “Don't forget to take a handful of our complimentary antibiotics
on your way out.”
Antibiotics resistance in United States
In 2008, the Infectious Disease Society of America (IDSA) confirmed that the United States and the rest of the
world are in the midst of an emerging crisis of antibiotic resistance for microbial pathogens [2]. Antibiotic
resistance is fueled by misuse and overuse of antibiotics. Bacteria become resistant to the very medicine
developed to treat and cure the infections they cause. Epidemic antibiotic resistance has been described in
numerous pathogens in varying contexts, including, but not limited to, a global pandemic of methicillin-resistant
Staphylococcus aureus (MRSA) infection [3], vancomycin-resistant enterococci (VRE), and a growing number of
additional pathogens that are developing resistance to many common antibiotics. The global spread of drug
resistance among common respiratory pathogens, including Streptococcus pneumonia [4-5] and Mycobacterium
tuberculosis [6], and epidemic increases in multidrug-resistant (and, increasingly, truly pan-resistant) Gramnegative bacilli [7], impacts clinicians practicing in every field of medicine. Given their scope of effect and
significant impact on morbidity and mortality, multidrug-resistant microbes are considered to be a substantial
threat to US public health and national security by the National Academy of Science's Institute of Medicine [8], the
federal Interagency Task Force on Antimicrobial Resistance (Interagency Task Force) [9], and the Infectious
Diseases Society of America (IDSA) [2]. In the United States, an estimated 23 × 106 kg of antibiotics are used
annually; about half of these are provided to people, and the rest are manufactured for agriculture and given to
animals including chickens, cows, pigs, and other livestock [10]. Livestock are fed antibiotics not because they are
infected, but for preventive purposes. These antibiotics are ingested by humans when they consume food. In
hospitals, antibiotics are generally administered parenterally, while in the community they are delivered mostly as
oral preparations. About 7 × 106 kg of antibiotics, chiefly penicillins and tetracyclines, are used as growth
promotants in food and animals [11]. Some 45 × 103 kg of tetracyclines and streptomycin are used as Golkar et al.
– Antibiotic resistance crisis J Infect Dev Ctries 2014; 8(2):129-136. 130 pesticides for agriculture; these are sprayed
on fruit trees in the southern and western United States [11]. While this last amount seems small compared with
overall antibiotic use, the geographical spread can be considerable. Some strains of Erwinia amylovora, the
bacterial target of these drugs, have become resistant to antibiotics. While the emergence of resistant bacteria in
agriculture is a small part of the overall global microbial resistance pool, it is an example of widespread antibiotic
use in which the environment of microorganisms is besieged with growth-inhibiting agents. The result is the
survival of those organisms that bear transposons and other mechanisms for selfpreservation, leaving
environmental microorganisms that are largely resistant [11]. Drug-resistant infections take a staggering toll in the
United States and across the globe. Just one organism, methicillin-resistant Staphylococcus aureus (MRSA), kills
more Americans every year than emphysema, HIV/AIDS, Parkinson’s disease, and homicide combined [12]. In
recent years, drugresistant forms of Neisseria gonorrhoeae, a causative agent of gonorrhea, the second most
commonly reported infectious disease in the United States, have begun to emerge. The National Institute of
Allergy and Infectious Diseases (NIAID) is investigating at several angles why the bacteria that cause gonorrhea are
becoming resistant to treatments, and is investigating new ways to treat and prevent the disease [13]. Currently,
the Centers for Disease Control and Prevention (CDC) recommends that most cases of gonorrhea in the United
States be treated with a combination of two drugs: a cephalosporin and a second antibiotic (e.g., azithromycin or
doxycycline). At this time of rising antibiotic resistance, it is important that more than one type of treatment be
available for infections such as gonorrhea, so that if the bacteria are resistant to one type of drug, other options
are available [14].
Phage history
In this pretext, we will not review the old literature that has been reviewed elsewhere [25,26]. To better
understand future prospects and phage application in modern medicine, however, some basic history is necessary.
The biblical Book of Kings relates how the prophet Elisha cured general Naaman’s disease by commanding him to
bathe seven times in the river Jordan. Since ancient times, there have been documented reports of river waters
having the ability to cure infectious diseases such as leprosy [27]. But, in 1896, the British bacteriologist Ernest
Hankin reported antibacterial activity against Vibrio cholerae, which he observed in the Ganges and Jumna rivers in
India. He suggested that an unidentified substance was responsible for this phenomenon and for limiting the
spread of cholera epidemics. Two years later, Gamaleya, the Russian bacteriologist, observed a similar
phenomenon while working with Bacillus subtilis [28]. From 1898 to 1918, others had similar observations of what
is thought to be the bacteriophage phenomenon. It was not until 1914, however, that another British
bacteriologist, Frederick Twort, advanced the hypothesis by proposing that it may have been due to, among other
possibilities, a virus. For various reasons, including financial difficulties, Twort did not pursue this finding [29]. A
French-Canadian microbiologist, Felix d’Herelle, first observed in 1910 the bacteriophage phenomenon while
studying microbiologic methods of controlling locusts in Mexico. In the lab, when he spread some cultures on agar,
he observed round zones without growth, which he called plaques, and asserted they were caused by viral
parasites. Six years later, he proposed the name “bacteriophage,” or bacteriumeater [29]. In 1917, d’Herelle began
testing his phage in human patients. Under the clinical supervision of Professor Victor-Henri Hutinel at the Hospital
des Enfants-Malades in Paris, he demonstrated the safety of his phages by ingesting them. The next day, he
demonstrated their efficacy by administering them to a 12-year-old boy with severe dysentery. The patient’s
symptoms ceased after a single treatment, and he made a complete recovery. Dr. d’Herelle’s anti-dysentery phage
was then administered to three additional patients, all of whom began to recover within 24 hours of treatment
[58]. In 1923, two physicians from Baylor University’s College of Medicine reported successful results from one of
their phage therapy trials conducted in United States, and concluded that “the bacteriophage holds enormous
possibilities as a new weapon for fighting infectious disease” [31].
Advantages of bacteriophage therapy
Phage therapy has several advantages over antibiotics. One of them is that phages are very specific and do not
harm the useful bacteria that live in and on the body.
Antibiotics, however, also attack harmless bacteria that make a living on us. This can, for instance, lead to severe
diarrhea, especially in patients with a weak immune system. Furthermore, phages attack also bacteria that are
resistant against antibiotics.
One advantage of phage therapy is that phages cannot grow without their target bacteria. Therefore, once they
have fulfilled their purpose and killed the pathogen, they too will die. Another advantage is they would be easy to
administer orally, intravenously, or topically.
Phages are also more host-specific than antibiotics, so it is less likely that there will be "collateral damage" relating
to killing normal and healthy gut flora along with the pathogen. This high specificity is also important because it
reduces the potential for secondary infections to develop (Inal 2003). If administered intravenously, it is easy for
the bacteriophages to spread rapidly throughout the body and they can also multiply on their own in vivo, which is
important because repeated doses are therefore unlikely to be needed. This proliferation of progeny in vivo would
also help in topical applications because they would be more able than antibiotics to infiltrate wounds and reach
more of the bacteria (Clark and March 2006). Phages also work quickly. One study of mice infected with
Vancomycin-resistant Enterococcus faecium found that if phage was injected 45 minutes after infection, 100% of
the mice were saved, and after just one day all were almost back to full health (Biswas et al 2002).
The specificity of phages makes them less toxic than some antibiotics such as Vancomycin used to treat MRSA
(Kutter et al 2012, Farber and Moellering 1983). Usually the levels of these antibiotics in the patient’s system are
closely monitored to prevent toxicity (Farber and Moellering 1983). As a result, dosage can’t be varied to handle
more resilient infections .These higher concentrations are occasionally required to eliminate certain bacterial
infections (Kutter et al 2012). Antibiotics also kill many of the bacteria that normally live on the human body
(normal flora) (Clark and March 2006). These bacteria provide us with specific vitamins and protect us from some
bacterial infections. Since phages are highly specific they will not harm our cells or the normal flora, which allows
us to use them in much higher concentrations . Since phages are viruses they are readily cleared out of our bodies
by our immune system. This prevents them from accumulating in our bodies and possibly causing any long lasting
toxicity, making phages even less toxic to our cells. This also presents the problem that phages can be prevented
from doing their job by being cleared out by our immune system. This is why the majority of phage cocktails are
applied directly to wounds so they won’t enter the blood stream and activate the immune system (Kutter et al
2012).
Health state in Vancomycin-resistant Enterococcus faecium-infected mice treated with heat-killed phage,
normal phage, and no phage over 4 days. 80% of mice treated with phage survived, while only 10% of
each of the other categories survived.
Antibiotics often have a problem reaching bacterial targets, especially if those targets have formed a biofilm that
the antibiotics cannot penetrate. Some bacteriophages synthesize enzymes that allow the breakdown of these
biofilms so that the interior cells can be reached and lysed (Clark and March 2006). Bacterial resistance to phages
is also less of a concern than resistance to antibiotics (Inal 2003). Even if resistance develops to a bacteriophage,
finding a phage that will still work against that bacteria would only take a couple of days. Phages mutate just as
much or more than bacteria, so they can better keep up with the bacteria evolving resistances. This ability to
continue to grow and multiply in the body is another advantage of phage therapy because while antibiotics are
broken down by the body and then excreted, bacteriophages are able to continue to keep their populations up and
even grow in number as long as the target bacteria is present. Another major advantage is that bacteriophages are
cheap to produce and the years of experimentation in Russia and Georgia have shown almost no side effects at all
(Inal 2003).
Also another advantages of phage therapy may arise as such,Phages are found throughout nature. This means that
it is easy to find new phages when bacteria become resistant to them, also phages can be genetically modified in
order to make up for some of their disadvantages.
However, there are also drawbacks of phage therapy. Probably the strongest is that, despite its long history, the
proponents of phage therapy never succeeded in producing formal proof of efficacy in human treatment. There
are many studies with positive results. But they all have one or the other weak point making definitive conclusions
difficult. There are several very good studies in animals that do show conclusively that phages can eradicate
infections in the bod
Clinical Use of Microphage Therapy:
After the advent and diffusion of antibiotics in Western Europe and the United States In 1950s and 1960s,
production of phages started and phages became used as both therapy and prophylaxis on a small scale. Smallscale productions of phages were produced by companies for different purposes [36].. Human phage therapy was
practiced in France since 1919 when d'Herelle treated children who were suffering from dysentery at the Hospital
des Enfants Malades in Paris with the phage he had isolated earlier from clinical samples, the children were
successfully treated [37]. . After this incident, the Pasteur Institute in France started to produce phages that work
against different pathogens such as Pseudomonas, Staphylococcus, Escherichia coli, and Serratia until 1974.
Thesephages were used clinically against wide variety of infections including skin infections, septicemia,
osteomyelitis, wound infections, urinary tract infections, and middle ear and sinus infections.
In 1979, the phage therapy was terminated. There are different possible causes of its termination; it could have
been terminated because of the thought that antibiotics would cure all infections in an easy way without the need
of knowing the exact causative pathogen in infected patients. Unfortunately, in developing countries, people can
buy a range of antibiotics without medical prescriptions.
Phages in the United States were used mainly in the production of human and animal vaccines. For example
Staphylococcal phage lysate (SPL) was used to protect animals and then was licensed for human therapeutic usage
after the safety trials were completed in 1959 [39]. It is administered by different routes: intranasal application by
aerosol, topically, orally, subcutaneously, and intravenously.
A rising interest in phage therapy started in the United States when the first studies about subcutaneous phage
application were published by the Michigan Department of Health. They reported the treatment of 208 patients
with chronic furunoculosis where 78% of patients had no recurring infections for at least six months after
treatments and only 3% of patients showed no improvement. There have been reports of success regarding
staphylococcus septicemia and meningitis. Some reports showed results of treating MRSA which can be
accomplished by local application for local infection or by systemic application – including intraperitoneally - for
systemic infections [41].
Other important infections are respiratory infections. Phage therapy can be used only in respiratory infections that
have a bacterial etiology. Klebsiella pneumoniae is another Gram negative bacteria that causes severe nosocomial
diseases such as septicemia, pneumonia, and urinary tract infection in immunocompromised individuals [43].
Community-acquired infection by invasive K. pneumoniae first emerged in Asia [44] and was then found worldwide
[45]. 80% of nosocomial infections caused by k.pneumonia are related to multidrug resistant strains.
Physicians categorized patients of respiratory infection depending on the presence of cystic fibrosis, and then
considered phage therapy in these patients. Previously, phage therapy has been reported to be successful in
treating pneumonia in six cancer patients [47].
A clinical trial was demonstrated to examine overall phage therapy efficacy that examined 607 patients, that all
failed conventional antibiotic therapy, and treated them with phage therapy. The results of the trial were good:
80% of patients recovered, 18% improved, and only 2% exhibited no changes. Also, no side effects of phages were
reported. Unfortunately, due to the regulatory pressure, the production of SPL was suspended for human therapy
in 1990 and now it is only used for veterinary applications [38].
Applications of phage therapy:
Delivery to targeted bacteria
When phages were first discovered and applied in a clinical setting for the treatment of diseases, they were directly
injected in the vicinity of the infection. Later mechanisms of delivery included topical administration 3 times a day, eye
drops, or orally before meals, but phages, unlike many antibiotic molecules, cannot diffuse across membranes and
therefore must have a method of delivery to the target cells. Some researchers believe that the best delivery
mechanism may lie in using other nonpathogenic species of bacteria to bring the phage to its pathogenic target [1].
Bacteriophage delivery modes have been categorized to “passive” and “active”, where passive delivery is achieved
by administering large doses of phage that exceed the bacteria present, and active delivery, involves extending the
phage’s life long enough to generate progeny cells which will then kill the bacterial cells.[2]
Effectiveness
Recent studies (in vitro and in vivo) of phage therapy have found that it can very helpful in killing bacteria.
One study by Huff et al (2003) found that treating chickens suffering from severe respiratory infections
cause by E. coli was very helpful in clearing up symptoms. They found that bacteriophage application was
most useful when applied shortly after the chickens had been exposed to the bacteria and that, if
treatment was initiatd early, multiple doses are better than a single dose, if however, treatment starts
later, there is no difference between single or multiple doses, but treatment is still very helpful, so it is
important to point out that phage therapy is the same as antibiotics in the necessity to catch the disease
early. Overall, this study found that phage therapy does seem to have a promising future and is very
effective as an alternative to antibiotics. [3]
One clinic in the United States has begun using phages in some limited applications, for example to treat
antibiotic-resistant infections. They have found that this method can be very effective. For example, one
woman had venous leg ulcers infected with multidrug-resistant pseudomonas. Within two weeks of phage
application the wounds began improving and within a few months the wound had drastically improved. [4]
Possible novel uses
Bacteriophages can be used in many applications related to therapy besides pathogens alone. For
example, it may be possible to use the products of phages rather than the whole viral organism. If
peptides such as lysins could be isolated from phages that do things like break down the peptidoglycan in
the bacterial cell wall, then it may be possible to administer the protein alone. This would be good
because many of these peptides are specific up to the subspecies level, and it has been found that the
mutations that the bacteria would require to resist this lysis would also kill the bacteria. [1]
There is a company in the United States, OmniLytics, devoted to making products from phages that can
be used in many contexts. They target bacteria in agriculture, they make products to improve food and
water safety, they treat industrial equipment, they experiment with pharmaceuticals, and they even
prepare for biohazard attacks such as those caused by biological weapons. This one company has been
able to take the concept of bacteriophages and apply it to a variety of areas to come up with some
specific and helpful innovations in many fields. For example, they received permission from the USDA to
treat live poultry with bacteriophage products before butchering to cut down on Salmonella contamination.
[5]
Conclusion
Bacterial resistance to antibiotics is a growing threat in our world. Though early studies of phage therapy did not
produce consistently favorable results, there is reason to believe that with the enhanced understanding that we have
of viral biology it is likely that bacteriophages can be a helpful therapeutic tool. Though it may be believed that phages
will not replace antibiotics, there is definite potential for their use in conjunction with antibiotics.
Bacteriophage resistance
In the last year’s bacterial resistance become a very dangerous problem for humanity, and it’s very important to
get a solution for this serious problem.
The scientists start the studying of bacteriophage (phage therapy).
Some of the bacterial species have been used for fermentation processes because the large quantities of
cultivation each day (2-5), but most fermentation industries have experienced problems with phages
contamination and this is a good point to use it as a phage therapy , but the main disadvantage it’s the risk of
bacteriophage resistance (6-10).
Now we will talk about some of bacteriophage resistance mechanisms:
A- “Preventing phage adsorption “:
The initial step for phage is to adsorb to host receptors, and the phage must recognize host cell components.
Unfortunately bacteria have range of barriers to prevent phage adsorption, and those barriers can be divided into
three categories:
1- “Blocking of phage receptors “.
2- “Production of extra cellular matrix “.
3- “Production of competitive inhibitors “.
1-
“Blocking of phage receptors “:
To limit phages in adsorb at bacterial cell surface and this can produced by conformational changes in their
structure.
Example: Staph aureus produce a cell wall with protein A ,and when bacteria produce less protein A (11), this
lead to improvement in phage adsorption because protein A responsible for blocking of the receptors (12).
Some host cells also use lipoproteins o inhibit phages, such as E-coli F+ strains.
Bordetella spp, alter their cell surface by phase variation, which is important for colonization and survival of
the bacteria (15) .
The expression of many bordetella adhesins and toxins is controlled by the two- component regulators system
(BvgAs).
Some of the toxins and adhesins under BvgAs control are expressed under Bvg+
There is one receptor which is expressed only on Bvg+, and that’s why Bvg+ higher efficiency of infection than
Bvg- (16) .
But still Bvg- able to have this infection without the receptor that responsible for it (pertactin autotransprter)
(Prn).
2-
“Production of extra cellular polymers “ :
Can protect the bacteria from any harsh conditions and providing a barrier for phages.
But some phages have he ability to recognize those polymers and sometimes degrade them by some enzymes
such as (21-25) :
a- The hydrolases. b- The lyases.
These viral enzymes are found either free from lysed bacterial cell, or bound to phage structure (22).
Example: alginates are exopolysaccharide produced by pseudomonas spp, azotobacter and some marine algae (22)
.
Phage resistance was observed for alginate producing azotobacter spp, but in pseudomonas spp, phage F116
which is responsible for this cell produces alginate lyase and degrades it (27).
3- “Production of competitive inhibitors “ :
Some molecules present in the bacterial environment can bind to the phage receptors and this receptor
become unavailable for phages.
Example: E-coli FhuA s an iron transporter, responsible for the entry of some phages such as (T1, T5).
Microcin J25 (antimicrobial) uses FhuA as receptor and competes with phage T5 for binding to the receptor.
And this microcin is produced under condition of nutrient depletion (41).
B “Preventing phage DNA entry “:
Super infection exclusion (Sie) system, are protein that block the entry of phage DNA into host cells.
Sie system in gram –ve ciliophage T4 has two Sie system encoded by:
1- Imm (immunity protein). , 2- Sp.
These systems inhibit the entry of DNA into the host cell, and they differ in their mechanism of action.
1- Imm change the confirmation of the injection site.
2- Sp inhibit the degradation of peptidoglycan by inhibit the activity of the T4 lysozyme.
Example: Sie A is found in the membrane of salmonella enterica subspp, carrieng lysogenic phage P22 (42).
Sie system in gram +ve, there is a very few example for this, such as lactococcus lactis a species used in milk
industrial processes (48-49).
C- “Cutting phage nucleic acid “:
Restriction-modification system, many bacterial genera posses (R-M) system.
The main function of (R-M) system is to protect the cell from invading DNA(53-54).
Conclusion:
Phages are now become as important ecological contributors in various environments(135).
In the last years, phage research becomes a very important as a phage therapy to combat ay bacteria, and to
combat them in food industries that use bacteria for fermentation.
That’s why it’s important to better understand the interactions between phages and their bacterial hosts and
how is the mechanism of action.
Finally, phage resistance mechanisms are often studied in a laboratory-controlled environment, one at time
and using a single phage-host model.
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1-Introduction references
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135
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Experimentalis. 2003. Volume 51. p. 237-244.
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biotechnology. 2006. Volume 24, Number 5. p. 212-218
[5] Walbeck, Alan. 2008. "OmniLytics Announces USDA/FSIS Allowance of Bacteriophage Treatment of Salmonella on Poultry".
http://www.omnilytics.com/news/news021.html
5-bacteriophage resistance references
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