CHAPTER THREE

PHAGE THERAPY: A POTENTIAL SOLUTION

3.1 Bacteriophages (Phages): An Introduction

- Nature and characteristics of phages

3.2 Historical Perspective

- Early use and decline of phage therapy

3.3 THE PHAGE LIFE CYCLE

According to Chanishvili (2012), Bacteriophage, a group of viruses that infect bacteria.

Bacteriophages were discovered individually by Frederick W. Twort in England (1915) and
Félix d'Hérelle in France (1917). D'Hérelle coined the term "phage", meaning
"bacteriophage", to describe the bactericidal power of an agent. Phages also infect single-
celled prokaryotes called archaea.

3.3.1 CHARACTERISTICS OF THE BACTERIOPHAGES

There are thousands of phages, and each phage can only infect a single species of bacteria or
archaea. Bacteriophages are divided into several viral families; examples include Inoviridae,
Microviridae, Rudiviridae, and Tectiviridae. Like all viruses, bacteriophages are simple
organisms that contain a core of genetic material (nucleic acid) surrounded by a protein
capsid. Nucleic acids can be DNA or RNA and can be double-stranded or single-stranded.
There are three basic structures of phages: an icosahedral (20-sided) head with a tail, an
icosahedral head without a tail, and a filamentous form (Suttle, 2007).

3.3.2 LIFE CYCLE OF PHAGE

During infection, phages attach to bacteria and insert their genetic material into the cells.
After that, phages undergo one of two life cycles: lytic (virulent) or lysogenic (simple). Lytic
phages recruit cellular machinery to produce phage components. When the cell breaks down
or breaks down, new phage particles are released. Lysogenic phages insert their nucleic acids
into the host cell's chromosomes and replicate as a single unit without destroying the cell.
Under certain conditions, lysogenic phages can induce the lytic cycle (Wittebole et al, 2014).

Other life cycles, including pseudolysogeny and chronic infection, also exist. In
pseudolysogeny, phages enter the cell but do not select the cellular replication machinery or
enter the genome. Pseudolisogeny occurs when host cells are exposed to adverse growth
conditions and appears to play an important role in phage survival by maintaining the phage's
genome until the growth of the host. In chronic diseases, new phage particles continue to
appear for a long time without actually killing the cells.
3.3.3 ROLE IN LABORATORY RESEARCH

Phages play an important role in laboratory research. The first groups studied were phages
types 1 (T1) to type 7 (T7). The T-even phages T2, T4, and T6 are used as models for virus
propagation studies. In 1952, Alfred Day Hershey and Martha Chase used phage T2 in a
famous experiment that showed that phage molecules require only nucleic acid.
Experimental results support the theory that DNA is genetic. Hirsch won the Nobel Prize in
Physiology or Medicine in 1969 for his work on bacteriophages. He awarded the award to
biologists Salvador Luria and Max Delbrück, who in 1943 conducted experiments with phage
T1 (variable test) that demonstrated phage resistance in bacteria. It is the result of normal
variation, not a direct response to environmental conditions. Some phages, such as lambda,
Mu, and M13, are used in recombinant DNA technology. Phage phiX174 was the first
organism to have its nucleotide sequence determined, a feat accomplished in 1977 by
Frederick Sanger and his colleagues (Ackermann, 2011).

In the 1980s, George P. Smith developed a technique called phage display that can produce
engineered proteins. The protein is produced by combining a foreign DNA segment or
produced with the phage III gene. Gene III encodes a protein that is displayed on the surface
of the phage virion. Thus, gene III is the fusion protein taken up by the phage and displayed
on the virion surface. Researchers can purify fusion phage cultures using developed
antibodies that recognize foreign protein fragments, and amplify foreign gene sequences for
further study. British biologist Gregory P. Winter later perfected phage display technology
for the development of human antibody proteins. The protein can be used to treat human
diseases with a lower risk of causing a serious allergic reaction than conventional medical
antibodies of animal origin. Adalimumab (Humira) is used to treat rheumatoid arthritis. It
was the first human antibody produced by phage display and was approved by the US Food
and Drug Administration (approved in 2002). Smith and Winter received the 2018 Nobel
Prize in Chemistry for their discoveries in phage display (Yoshikawa, 2020).

Phage expression Shortly after this discovery, Tewater and de Herer began using phages to
treat bacterial diseases in humans, such as the Black Death and cholera. Phage therapy was
unsuccessful and was largely abandoned after the discovery of antibiotics in the 1940s.
However, with the rise of antibiotic-resistant bacteria, the therapeutic potential of
bacteriophages (Hicks et al, 2016).

3.4 MECHANISMS OF PHAGE ACTION

Bacteria evade phage infections in different ways. Here, we classify different resistance
mechanisms in three main categories:

Receptor adaptations: random mutations or phenotypical variations in bacteria that result in

decreased phage adsorption (Fig. 3.1).
Figure 3.1: Host adaptations leading to phage resistance

(A) Point mutations can lead to a loss or modification of the phage receptors (green
rectangles), or to downregulation of their expression. (B) Receptor masking proteins like
TraT of Escherichia coli (pink) can bind to the surface-exposed regions of phage receptors,
making them unavailable for the phages. (C)Outer-membrane vesicles (OMVs) presenting
phage receptors act as decoys to prevent the phages from encountering the bacteria. (D) An
increase in the production of extracellular matrix (light green) leads to phage receptors
being physically hidden. (E) Phase variation occurs through three mechanisms: site-specific
recombination, slipped-strand mispairing and epigenetic modifications. It can regulate the
bacterial phenotype, including the expression of surface proteins like phage receptors.

Host defense systems: molecular pathways that have specifically evolved in bacteria to
prevent or suppress phage infections (Fig. 3.2).
Figure 3.2: Host phage defense systems

(A) Multiple defense systems act via nucleic acid interference. R-M systems are generally
composed of an MTase that methylates endogenous DNA to distinguish it from exogenous
DNA, and of an REase that cleaves the exogenous, non-methylated DNA. DISARM interacts
with phage DNA to prevent its circularization, thereby blocking its replication or lysogeny.
BREX or Ago systems interact with phage DNA and prevent it from replicating without
necessarily cleaving it. CRISPR-Cas systems are known as the adaptive immune system of
bacteria. The CRISPR array contains sequences of foreign origin that can be transcribed and
processed to act as a guide for the Cas endonuclease, which recognizes and cleaves said
sequences upon reentry into the bacteria. (B) Abortive infection comprises a series of
mechanisms that lead to bacterial cell suicide. An example in which this can happen is
through an imbalance in the concentration of toxins and antitoxins in a cell. Another example
is through the action of effector proteins that might get activated directly, like in the case of
retrons, or via second messengers, like in the case of CBASS or Thoeris. These effector
proteins can lead to cell death in several ways, for instance through inner membrane
degradation (CBASS) or through NAD depletion (Thoeris). (C) Bacteria can produce
secondary metabolites such as daunorubicin (depicted) that intercalate phage DNA and
prevent it from circularizing and replicating. (D) Analysis of genetic defense islands has
recently led to the discovery of a series of defense systems that are yet to be fully
characterized. These include: Hachiman, Shedu, Gabija, Septu, Lamassu, Zorya, Kiwa,
Druantia, Wadjet, RADAR, DRTs, AVAST and pVips, among others.
Phage-derived phage defense systems: molecular pathways encoded by phages to compete
with other phages to the benefit of the host (Fig. 3.3).

Figure 3.3: Phage-derived defense systems

(A) Superinfection exclusion systems (Sie) are encoded by phages to prevent other phages
from infecting their host. Some phages like T5 produce proteins that mask their receptor and
make it inaccessible. Other phages, especially prophages, encode membrane-associated
proteins that interact with the phage receptor, blocking the DNA entry channel, triggering a
conformational change or inhibiting the invading phage's enzymes. (B) Prophages like
Panchino of Mycobacteriumsmegmatis can confer resistance to their hosts through the
expression of R-M systems or DNA-binding repressor proteins that target the DNA of newly
infecting phages. Other prophage-encoded systems, like RexA-RexB or the newly
characterized PARIS, can trigger an Abi response upon sensing an invasion by a new phage.

3.4.1 RECEPTOR ADAPTATION LEADING TO PHAGE RESISTANCE

In natural environments, bacteria are under constant selection pressure, which forces bacteria
and phages to develop defense systems and engage in an arms race to combat them. Arms
races are characterized by high mutation rates and horizontal gene transfer, leading to rapid
evolution of genetic traits and genetic diversity (Laxminarayan, 2013). Mutations that cause
changes on the cell surface can inhibit phage adsorption and are therefore directly beneficial
to the host. Bacteria can present a barrier to phage adsorption by reducing the availability of
receptors for phage binding. Acquiring point mutations in the genome (Figure 1A) is
probably the easiest way for bacteria to acquire complete resistance to phages. Indeed,
mutation of receptor genes or their regulation has been a common method to identify phage
receptors. These mutations often occur during phage attack and can cause loss or reduction of
gene expression of a particular receptor or changes in its structure. For example, E. coli
mutates tolC and lipopolysaccharide (LPS) genes to resist infection by phage U136B.
Similarly, Acinetobacter baumannii mutates genes involved in capsular polysaccharide
biosynthesis to avoid infection by phages øFG02 and øCO01. In Listeria monocytogenes,
loss or deficiency of wall teichoic acid rhamnosylation results in resistance to various phages
and also leads to serotype diversification. Other proteins involved in phage adsorption and
DNA injection, such as Enterococcus faecalis phage infectivity protein (PIPEF), can also
mutate in response to phage attack (Zhang et al. 2009).

Bacteria can also block phage attachment by producing proteins that mask or block phage
receptors on the cell surface. An example of this is the protein TraT, which is encoded by the
F plasmid. It localizes to the outer cell membrane and binds to the surface exposed region of
the E. coli outer membrane protein OmpA. This makes this common phage receptor
inaccessible to phage binding. Masking molecules such as lipoproteins that bind to phage
receptors can also be produced by bacteria under stressful conditions and released during
bacterial lysis. Some bacteria produce and release OMVs that act as cellular decoys,
capturing and inactivating phages. Another mechanism that prevents phages from reaching
receptors is the upregulation of the production of extracellular matrix, which is typically
composed of polysaccharides, proteins, lipids, and extracellular DNA. Must be protected
from phage adsorption (Hanlon). In Lactococcus lactis, plasmids encoding exopolysaccharide
biosynthetic genes can also confer protection against phages. Additionally, reversible changes
in the control of gene expression, a phenomenon known as phase fluctuations, can cause a
decrease in receptor availability. These changes can be mediated by site-specific
recombination, in which the inversion of DNA segments in the promoter or regulatory region
of a gene turns its expression on or off. This is evidenced by the development of flagella in
Salmonella spp. Illustrated. and Escherichia coli fimbriae. Other receptors, such as the
meningococcal outer membrane protein Opc and the subunits of the Bordetella pertussis
fimbriae is controlled by the slip strand mismatch. H. It is due to programmed mutations that
occur in defined regions during DNA recombination. Epigenetic modifications, such as
changes in the methylation pattern of DNA sequences, also control the expression of phage
receptors, such as the O antigen chain of the LPS of Salmonella enterica (CDC, 2017).

All of these changes act directly on the phage receptor and reduce the likelihood of phage
adsorption. However, modification of surface elements may involve trade-offs with host
bacterial fitness in the form of host toxicity or reduced survivability, thereby Possibilities are
limited. Therefore, more specific defense systems targeting phages within host cells are also
required, especially in the context of complex microbial communities (WHO, 2017).

3.4.2 HOST PHAGE DEFENSE SYSTEM

Bacteria have developed defense systems that protect them from mobile genetic elements
such as phages. Many of them are concentrated in genomic regions known as defense
islands, offering opportunities to discover new defense systems by analyzing genetic regions
near other known defense systems. Offers. Such strategies have significantly and rapidly
expanded the known bacterial reservoir for protection against phage infection. We highlight
the various phage defense systems that have been identified, including those that act on viral
nucleic acids and those that cause incomplete infection of the host.

NUCLEIC ACID INTERFERENCE

The ability to manipulate viral nucleic acids is a common strategy used by hosts to limit
phage entry and proliferation.

One of the most widely known and oldest examples of phage defense systems is the so-called
restriction-modification (R-M) system, which acts on phages with DNA genomes. In the R-M
system, methyltransferases (MTases) methylate endogenous DNA at specific sites, protecting
it from cleavage by restriction endonucleases (REases). REase recognizes foreign,
unmodified DNA and stores it within, near, or away from the recognition site. Split (Boucher
et al, 2009).

There are four classical types of R-M systems (I–IV), classified according to the
characteristics of specific components. Type I R-M systems consist of a protein complex of
three subunits with different activities: M (MTase), R (REase), and S (specificity) subunits.
The S subunit determines the target sequence specificity of both methylation and restriction
by protein complexes. Common type II R-M systems include MTase and REase, which
function independently as separate proteins. These are the main source of hundreds of
commercially available restriction endonucleases used in molecular cloning. The type III R-
M system also expresses two independent MTase and REase proteins, but these function as
a complex. The type IV R-M system does not contain MTase and is thought to have evolved
in response to some phages evading the type I-III R-M system by modifying their genomes to
circumvent evasion restrictions. I am. Type IV systems overcome this counterattack by
limiting the modified DNA of phages while leaving bacterial DNA unmethylated.
Interestingly, MTases tend to be more conserved than REases, as the latter evolve rapidly to
keep pace with mutations in the phage genome (Lurepke et al, 2017).

R-M systems typically provide epigenetic marks on the nucleobases. However, similar
systems have been described that modify sugar-phosphate backbones by introducing
phosphorothioates (replacing non-bridging oxygens with sulfur). The Dnd system functions
through double-stranded phosphorothioation of endogenous DNA by the protein DndABCDE
and restriction of foreign unmodified DNA by DndFGH. The Ssp protein SspABCD also
alters the host genome through phosphorothioates, but only one of the two DNA strands is
altered. This activity was coupled with the activity of SspE, which requires recognition of
SspABCD to introduce nicks into foreign DNA, or with the activity of SspFGH, which
indiscriminately damages non-phosphorothioate DNA and inhibits its replication (CDCP,
2013).

Our knowledge of R-M-related defense systems continues to expand as more systems are
discovered through analysis of bacterial genomes. One example is the DISARM system
(Defense Island System Related to Restrictions and Modifications) include MTases (adenine
MTase DrmMI and/or cytosine MTase DrmMII) and proteins with domains of unknown
activity or function (DrmB, DrmE) belongs. Although the exact mechanism of action of
DISARM is not yet clear, it has been shown that methylation of host DNA is involved in
distinguishing between self and non-self, and that it inhibits DNA replication by preventing
phage DNA circularization. , it is clear that lysogeny inhibits infection early. stage. It is also
hypothesized that DISARM can cooperate with various R-M elements to achieve synergistic
effects against phage infection. Bacteriophage exclusion (BREX) defense systems also target
phage DNA upon entry into host cells. Similar to the R-M system, BREX methylates host
DNA to distinguish it from foreign DNA. However, BREX does not appear to degrade
unmethylated phage DNA, but instead appears to prevent phage DNA replication without
cleavage. Although methylated or glycosylated phage DNA is not susceptible to BREX,
deletion of the methylase gene in this system has no negative effect on the bacteria (Donlan,
2009).

3.5 ADVANTAGES AND POTENTIAL OF PHAGE THERAPY

In Bourdin et al (2014), advantages of phage therapy over the use of chemical antibiotics can
be framed in terms of phage properties. In this section we consider those properties that, in
our opinion, can contribute substantially to phage therapy utility.

BACTERICIDAL AGENTS.

Bacteria that have been successfully infected by obligately lytic phages are unable to regain
their viability. By contrast, certain antibiotics are bacteriostatic, such as tetracycline, and as a
consequence may more readily permit bacterial evolution towards resistance.

AUTO “DOSING”.

Phages during the bacterial-killing process are capable of increasing in number specifically
where hosts are located,Citation5 though with some limitations such as dependence on
relatively high bacterial densities. We call this auto “dosing” because the phages themselves
contribute to establishing the phage dose.

LOW INHERENT TOXICITY.

Since phages consist mostly of nucleic acids and proteins, they are inherently nontoxic.
However, phages can interact with immune systems, at least potentially resulting in harmful
immune responses, though there is little evidence that this actually is a concern during phage
treatment. Nonetheless, it can be imperative for certain phage therapy protocols to use highly
purified phage preparations to prevent anaphylactic responses to bacterial components, such
as the endotoxins that can be found in crude phage lysates. Phages similarly can release
bacterial components while killing bacteria in situ, a property associated with lysis that also
can result from the application of cell-wall disrupting antibiotics.
MINIMAL DISRUPTION OF NORMAL FLORA.

Owing to their host specificity—which can range from an ability to infect only a few strains
of a bacterial species to, more rarely, a capacity to infect more than one relatively closely
related bacterial genus—phages only minimally impact health-protecting normal flora
bacteria. By contrast, many chemical antibiotics, which tend to have broader spectrums of
activity, are prone to inducing superinfections, such as antibiotic-associated Clostridium
difficile colitis or Candida albicans yeast infections. The historical bias towards developing
only broader spectrum antibiotics, however, may be changing.

NARROWER POTENTIAL FOR INDUCING RESISTANCE.

The relatively narrow host range exhibited by most phages limits the number of bacterial
types with which selection for specific phage-resistance mechanisms can occur. This
contrasts with the substantial fraction of bacteria that can be affected by most chemical
antibiotics.Citation5 In addition, some mutations to resistance negatively impact bacterial
fitness or virulence due to loss of pathogenicity-related phage receptors.

LACK OF CROSS-RESISTANCE WITH ANTIBIOTICS.

Because phages infect and kill using mechanisms that differ from those of antibiotics,
specific antibiotic resistance mechanisms do not translate into mechanisms of phage
resistance. Phages consequently can be readily employed to treat antibiotic-resistant
infections such as against multi-drug-resistance Staphylococcus aureus.

RAPID DISCOVERY.

Phages against many pathogenic bacteria are easily discovered, often from sewage and other
waste materials that contain high bacterial concentrations. Isolation can be more technically
demanding, however, if host bacteria themselves are difficult to culture and bacteria may
differ in terms of the number of phage types to which they are susceptible. Unlike antibiotics,
which can be toxic, phages that display little or no toxicity can be isolated against most target
bacteria.

FORMULATION AND APPLICATION VERSATILITY.

Phages, like antibiotics, can be versatile in terms of formulation development, such as being
combined with certain antibiotics. They are also versatile in application form, as liquids,
creams, impregnated into solids, etc., in addition to being suitable for most routes of
administration. Different phages furthermore can be mixed as cocktails to broaden their
properties, typically resulting in a collectively greater antibacterial spectrum of activity.

BIOFILM CLEARANCE.

Biofilms tend to be substantially more resistant to antibiotics than planktonic bacteria.

Phages, however, have a demonstrated ability to clear at least some biofilms, perhaps owing
to a potential to actively penetrate their way into biofilms by lysing one bacterial layer at a
time, or due to the display of biofilm exopolymer-degrading depolymerases.
 CHAPTER FOUR

CURRENT RESEARCH AND APPLICATIONS

4.1 RECENT ADVANCEMENTS IN PHAGE THERAPY

As noted by Wahida et al (2016), Phages are natural killers of bacteria and offer great
potential in combating the growing threat of MDR strains. Although phage cocktails have
proven to be an optimal solution for clinical use, further cocktail combinations need to be
explored to identify the most effective cocktails and further elucidate the mechanisms of
combination. In addition to phage therapy, endolysins and depolymerases may also be
involved in the synthesis of antimicrobial peptides or other antimicrobial agents or used to
remove A. baumannii biofilms from medical devices. However, due to the high specificity of
depolymerases such as phages, their applications have been limited.

Thanks to the widespread use and application of metagenomic sequencing technology,

numerous novel phages and large phage genomes have been discovered, adding new vitality
to the field of phage research. Further analysis of the unknown functional proteins of the A.
baumannii phage and promoting the development of phage-derived enzyme preparations are
expected to improve the application efficacy and scope of the phage. The rapid emergence of
phage resistance in bacteria has made the use of phage therapy essential. Therefore, basic
research is required to reveal the detailed interaction mechanisms of A. baumannii phages,
including the characteristics of phage host receptors and the molecular mechanisms of his A.
baumannii phages. This may also provide important theoretical support for genetic
modification and artificial synthesis of phages in clinical applications (Delbrück, 1940).

Combinations of phages and other antimicrobial agents also show promise for the treatment
of A. baumannii, and further experimental studies are needed to determine the optimal
combination regimen and application strategy. The combination of phages and
photosensitizers is an interesting direction and may have great practical potential in
combating multidrug-resistant A. baumannii and in biofilm ablation. The safety of an
individual phage preparation depends not only on the safety of the phage itself, but also on
the method of its production. Therefore, standardization of phage preparation methods is
critical, as purification of phages from bacterial hosts can lead to unintended release of
bacterial toxins, such as endotoxins and/or exotoxins. Several studies have highlighted the
importance of standardizing the production of phage preparations and are actively promoting
related research advances for therapeutic use. Since phage encapsulation is advantageous for
delivery in clinical applications and to improve the efficiency of phage therapy, more
attention should be paid to this approach in the treatment of A. baumannii infections.
Innovative applications of novel materials in phage combination therapy or phage
encapsulation may be promising directions for the future (Weinbauer, 2004).

As an ancient and effective biological control method, phage therapy has become a hot
research area around the world due to the continued emergence of MDR bacteria, which
represents a setback in the development of new antibiotics.
4.2 CASE STUDIES

A research group led by scientists from the University of Pittsburgh and the University of
California, San Diego, presented 20 new case studies on the use of phage therapy, showing
success in in more than half of the patients. It is said to be the most widely read collection of
phage therapy case studies to date, providing information on phage therapy for disease while
laying the foundation for future clinical trials.

Graham Hartfull, the Eberly Family Professor of Bioengineering at the Kenneth P. Dietrich
College of Arts and Sciences at the University of Pittsburgh, said, "Some of these results are
dramatic, while others are difficult. it is increasingly recognized that phages provide
beneficial outcomes for patients who have no other options."

The team published the paper "Bacteriophage therapy for mycobacterial infections: the use of
phages in twenty patients with mycobacteriosis-prevention" in the journal Clinical Infectious
Diseases. “Infections caused by nontuberculous mycobacteria (NTM), particularly
Mycobacterium abscessus, are increasingly common in patients with cystic fibrosis and
chronic bronchiectasis. Treatment is difficult due to strong antibiotic resistance. Phage
therapy may be a new approach. Few lytically active phages are available, and phage
susceptibility varies among M. abscessus isolates, requiring individual phage identification,"
the researchers wrote. "Mycobacteria isolated from 200 clinical and symptomatic patients
were examined for phage susceptibility. One or more lytic phages were identified for 55
isolates. Based on the work charity, 20 patients will be injected with phages by injection,
nebulization, or both, and patients will be monitored for adverse effects, clinical and
microbiological response, the emergence of phage resistance, and elimination of phages in
serum, sputum, or bronchoalveolar lavage fluid.

"No treatment-related adverse events were observed in any patient, regardless of the
pathogen, phage, or delivery method used. A good clinical or microbiological response was
observed in all patients. Antibodies were detected in the serum of eight patients after the start
of intravenous phage administration, which may have contributed to the lack of treatment
response in four patients but was not significantly associated to the side effects on the rest of
the patients. Eleven patients received only one phage therapy, and phage inhibition was not
detected in these patients.

"Treatment of mycobacterial infections is challenging due to the limited repertoire of

effective phages, but good clinical results in patients with no treatment options support the
continued development of adjuvant phage therapy for some mycobacterial infection."
Figure 4.1: A closed bacteriophage virus attacking a bacterial cell 3D illustration

Every patient included in the study was infected with one or more types of mycobacteria, a
type of bacteria that can be fatal and difficult to treat in people with weakened immune
systems or cystic fibrosis. In 2019, a team led by Hatfull successfully used phages to treat
one of these diseases for the first time.

"This is a real nightmare for doctors: It's not as common as other infections, but it's one of
the hardest to treat with antibiotics," Hartfull said. "Especially if you take these antibiotics for
a long time, they're toxic or they're not good."

Last month, researchers from the University of Pittsburgh, in collaboration with colleagues at
the National Jewish Health and Harvard University, participated in two new case studies
showing success to treat the sick. This new paper includes colleagues from 20 institutions.

"These are incredibly brave doctors who jumped off the cliff to develop experimental
treatments to try to help patients who had no other options," Hatfull said. "Every
collaboration is an opportunity to advance the field."

The team found that the treatment was successful in 11 of 20 cases by looking at the patient's
health and the patient's samples still showed signs of mycobacterial infection. The patients
did not report any harm to the treatment. The results of treatment in five patients were
indeterminate, and four patients did not improve. Hatfull says this uncertainty is key to
making this drug available to more patients. "In some ways, this is a very good case," he said.
"It's important to understand why it doesn't work."
4.2.1 UNEXPECTED PATTERNS

Several surprising patterns emerge from these case studies. In 11 cases, the researchers did
not find more than one phage that caused the patient's disease, although the standard
procedure is to inject different types of viruses so that the bacteria are less likely to develop
resistance. "If you had asked me three years ago if this was a good idea, I would have been
very angry," Hartfull continued. "But we didn't see resistance, even with a single phage, and
we didn't see resistance leading to no treatment."

In addition, the research team also found that some patients' immune systems attacked the
virus, but sometimes their immune systems were ineffective. In some cases, treatment is
successful despite an allergic reaction. Hatfull said the study provides an encouraging picture
of this therapy and opens up the possibility of new phage therapies that doctors can use to
increase the chances of successful treatment.

Hatfull said that in addition to the importance of this research for patients with mycobacterial
infection, it also represents a major advance in the general field of phage therapy, adding that
some were concerned that researchers only publish cases of phage therapy. successful. "It's
an ongoing series of case studies where we don't pick a bond, but it's a much easier way to
see what works and what doesn't," Hartfull said. "This medicine is much safer.

"We haven't found a way to detect or engineer phages that infect all types of bacteria in these
patients. This is one of the biggest challenges in the future."

4.3 CHALLENGES AND LIMITATIONS

4.3.1 DISADVANTAGEOUS CHARACTERISTICS OF PHAGES

The spectrum of phage lysis is very narrow due to its high specificity. Phages usually only
target certain types of bacteria, while some bacteria target only certain species, so they cannot
target all pathogenic strains of the same bacterial species. Phages can be used to treat
infections caused by a single bacterium, but clinical cases are often caused by multiple
pathogenic bacteria. Therefore, it is still difficult for some phages to achieve the desired
therapeutic effect. Lysogeny refers to the inability of some lysogenic phages to lyse host
bacteria after attachment to host bacteria, and to inhibit the lysis of other phages of their host
bacteria. In lysogeny, the human genome is converted to host DNA in a plasmid-episomal
form or after integration into the bacterial chromosome. In addition, the more important issue
is that lysogenic phages can transmit toxins and antibiotic resistance genes to bacteria.

Compared to protein drugs that can be assessed for activity and purity based on specific
antibody markers, the structure of PT preparations is more complex, containing proteins and
nucleic acids. Therefore, it is difficult to assess its quality and strength.

4.3.2 LACK OF APPROPRIATE POLICIES

There are no policies or regulations for the clinical implementation of PT. Appropriate
regulatory standards will contribute to increasing awareness of this effective treatment.
Verbeek et al. The views of European stakeholders are analyzed in detail and the need to
adapt the legal framework to include HEIs is also discussed. An important consideration is
whether PT development will be conducted on an industrial scale or on a hospital-based
scale. They proposed a new European legal framework for HEIs. In addition, there are no
clear standards for phage isolation and purification, so the requirements for phage isolation
vary. There is no standard method for the clinical treatment of phages.

4.3.3 RESISTANCE OF BACTERIA TO PHAGES

With the emergence of phage-resistant strains, various studies have shown that if the same
phage is used continuously for a long time, bacteria will also develop phage-resistant strains
during natural selection. . This is part of a series of anti-phage strategies that have long been
developed by bacteria, including adsorption resistance, immune modification systems,
resistance to injection, infection, resistance to superinfection, and clustering and
incorporating short palindromic information linked by CRISPR (Clustered Regularly
Interspaced Short Palindromic Repeat System-CRISPR) CRISPR-Cas). Blocking entry
reduces the interaction between phage and bacteria. In bacterial infections, phages and
bacteria are killed. CRISPR-Cas is part of the adaptive immune system, which provides
bacteria and archaea with adaptive resistance to foreign invaders such as plasmids and
phages. CRISPR and Cas proteins combine to form a widespread system in bacteria and
archaea that disrupts foreign nucleic acids. The CRISPR-Cas system works in at least two
stages: the adaptation stage, where cells acquire new spacer sequences from foreign DNA;
and in the intervention phase, recently spaced arrays have been used to detect and break
down invasive nucleic acids. CRISPR-Cas systems contribute to the continuous evolution of
phages and bacteria by adding or deleting sites in host cells and modifying or deleting phage
genes.

4.3.4 LACK OF PHAGE PHARMACOKINETIC DATA

PT preparation is difficult in comparison. The definition of the measurement is not yet clear.
Furthermore, the administration method and dosage of PT directly influence its effects,
making the clinical application of PT difficult. Since phages are mainly protein and DNA or
RNA, they are easily degraded when they come into contact with humans, such as in the
stomach and liver, and when they come into contact with the animal's immune system.
Related pharmacokinetic studies showed that a quarter of the injected phage was retained 36
hours after treatment, but the effective concentration was diluted by body fluids. Oral
administration is the preferred route of administration for humans and animals. In addition,
this method is simple and convenient, and is less immunogenic than other methods of
administration. During oral administration, phage particles cross the gastric, intestinal, and
intestinal mucosa before reaching the systemic circulation. Therefore, the gastrointestinal
system is considered the main barrier to entry of phage into tissues. Additionally, the
mammalian circulatory system can eliminate phages from the blood, making it difficult to
maintain sufficient phage concentrations to kill bacteria.
4.3.5 COMMUNICATE WITH THE BODY

When phages degrade bacteria, they release bacterial toxins, such as endotoxins, which cause
more bacterial infections. Sometimes, a purulent infection occurs. Related experiments have
shown that oral administration of phage mixtures to mice increases intestinal permeability
and endotoxemia.

Foreign proteins carried by phages can interfere with human or animal immune responses.
This reaction is unusual because phages have been shown to be safe. These reactions are
immune responses to proteins associated with the phage virion.

Several studies have reported that PT can treat various diseases, but there are no data from
double-blind randomized clinical trials.

4.4 FUTURE DIRECTIONS

Medicines that use bacteriophages can effectively kill a variety of bacterial infections in
humans, especially when combined with antibiotics. One possibility is the treatment of
infected epidermal wounds, which often occur in patients with allergies and diabetes. Phage
therapy can be administered as a topical solution, injected into or around a wound, using a
biodegradable biopolymer coating with extended release capabilities. Pathogens
(Mycobacterium spp.) also need to be approached to develop effective methods for phage
delivery to mammalian cells, although pathogens with accessible extracellular life may be
more susceptible to the phage target. An example is Mycobacterium ulcerans, Trigo et al.
Phage therapy has been shown to be effective in targeting mouse footplate models. 68 For
antibiotic resistant strains, Golkar et al. described the efficacy of bacteriophage PS5 against
multi-resistant Pseudomonas aeruginosa strains in mouse models with particular attention to
the efficacy of this treatment. Mice were injected with lytic PS5 twice, once more than 30
minutes and hours after bacterial infection, followed by oral doses of PS5 via drinking water.
The wound patterns in the phage group had all healed after 6 days, compared to the
remaining open wounds on the backs of untreated infected animals. Intraperitoneal and oral
administrations have been established as effective treatment options.

The mucosa is very attractive for treating bacterial colonization and phage therapy, due to its
ability to infect. Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa,
and Staphylococcus aureus have been extensively studied in phage therapy for respiratory,
gastrointestinal, and cancer. However, there are still many pathogens that should be
investigated in phage therapy, including upper respiratory infections caused by Haemophilus
influenzae, diphtheriae, and Porphyromonas species, lower respiratory infections caused by
Helicobacter pylori, Mycobacterium simiae, or Nocardia species. Species that cause
pneumonia, including Mycoplasma pneumoniae, Fluorobacter botzma, or Ureaplasma
urealyticum, provide important populations for the use of phage therapy. The most effective
treatment for respiratory infections is by inhalation, directly into the airways and minimizing
exposure to other substances. Infectious system phages. For intestinal infections, phage
encapsulation or encapsulation is important to avoid exposure to low pH and digestive
enzymes during oral administration. Effective treatment of cattle containing E. coli using
phage O157:H7 has been shown to be effective against E. coli. Phage therapy also has an
advantage over traditional antibiotic treatment by reducing the damage to the intestinal flora.
Treatment of infectious diseases and further research, as there are no lytic phages for
Legionella pneumophila, Proteus pennei, Ryeella, and Citrobacter kossei. In addition, phage
therapy has great potential for treating the causes of sexually transmitted diseases, including
Haemophilus ducreyi (chancroid), Chlamydia trachomatis (chlamydia), Neisseria
gonorrhoeae (gonorrhea), Treponema pallidum (syphilis), and the granulomatous Gram-
negative bacterium Lebsiella pneumoniaerium. (Granuloma inguinale).

Finally, phage therapy shows potential in treating life-threatening systemic diseases that can
easily develop from local infections such as pneumonia, urinary tract infections, or cancer,
and skin disease. Bacteria of particular concern include: Francisella spp., Leptospira
interrogans, Brucella canis, Ehrlichia spp., Rexella sp., Treponema spp., Anaerobic
Peptostreptococcus, Clostridium tentativecum, and Clostridium botulinum. Treatment by
injection into the bloodstream can eliminate the disease from the bloodstream, but
transdermal administration can be another way of treatment. For systemic therapy, this can
reduce the risk of host tissue damage from chemical antibiotic treatment, as phages infect
specific bacteria and thus help to protect the integrity of the patient's tissue and its native
microbiota. Phage therapy has advantages over current bacterial treatment options,
particularly its flexibility, specificity, and lack of host resistance. Methods for breeding phage
populations used for screening must be developed and refined continuously in a consistent
and reproducible manner, and ongoing stability, characterization, and testing procedures must
be completed to good medicine. The issues that need to be addressed are the lack of quality
rules governing the therapeutic potential of phages, as there is a need for extensive testing to
ensure the safety and efficacy of phages as antibacterial drugs, other prescribed drugs.
 CHAPTER FIVE

CONCLUSION AND FUTURE PERSPECTIVES

5.1 CONCLUSION

Bacteriophages are bacteria-killers and show great potential against the virulent threat of
MDR strains. Phage mixtures have been identified as a promising solution for clinical
applications. At the same time, other combinations should be investigated to determine the
best therapeutic effect and to reduce drug resistance. Also explain the compounding process.
In addition to phage therapy, endolysins and depolymerases can also be added to the
synthesis of antimicrobial peptides and other antibacterial drugs, each of which is used to
remove Acinetobacter baumannii biofilms from equipment Medicine. However, the high
specificity of bacteriophage-like depolymerases limits their application.

With the popularity and application of metagenomic sequencing technology, many new and
important phage species have been discovered, bringing new life to the field of phage
research. Further analysis of unknown functional proteins of Acinetobacter baumannii phages
and promoting the development of phage-derived enzyme preparations will further impact the
application and coverage of phages. The rapid development of bacterial phage resistance is
an important application of phage therapy. Therefore, more research is needed to show the
interaction mechanism of the A. baumannii phage in detail, including the features of the
phage host and the molecular structure of the A. baumannii phage. Research on the
resistance of Acinetobacter baumannii to phages can also provide important theoretical
support for the clinical application of genetic modification and phage synthesis.

The combination of bacteriophages with other antibacterial drugs also holds promise for the
treatment of Acinetobacter baumannii, so further experimental studies are needed to establish
effective combination programs and strategic use. The combination of phages and
photosensitizers is an interesting approach that may have great potential in the fight against
multidrug-resistant Acinetobacter baumannii and work for biofilm destruction. The safety of
a single phage preparation depends not only on the safety of the phage itself, but also on the
method of production. Thus, phage preparation techniques are important, as purification of
phages from the bacterial host can result in the harmful release of bacterial toxins such as
endotoxins and/or exotoxins. Several studies show the importance of standardization of phage
preparations and the potential to promote the application of medically relevant research
advances. As phage delivery methods facilitate delivery in clinical applications and improve
the efficiency of phage therapy, more attention should be paid to the treatment of A.
baumannii infections. The innovative application of new materials in phage combination
therapy and phage synthesis may be promising avenues for the future.

An ancient and effective method of biological control, phage therapy has become a center of
research worldwide due to the continuous emergence of multidrug-resistant bacteria, leading
to the prevention of development of new antibiotics.
5.2 THE PROMISE OF PHAGE THERAPY

The world today relies on antibiotics to fight bacterial infections, but their use has given rise
to new drug-resistant "superbugs" that are now evolving to fight against most antibiotics.
More than 1.27 million people died worldwide in 2019, according to the US Centers for
Disease Control and Prevention. Furthermore, antibodies are not specific. These drugs are not
only anti-bacterial, but they also disrupt the beneficial microbes in our guts, reducing the
diversity of microbes, and it can take months to settle down. returning to a normal
microbiome after antibiotic treatment. Currently, we can still treat most of the bacteria that
are resistant to drugs, but this is not always the case, so other methods are needed. A long-
standing alternative is phage therapy.

Although we have long known the limitations of current antibiotics, little effort has been
done to find new antibiotics and further research. There is a call for the development of new
antibiotics, but major pharmaceutical companies are reluctant to fund early-stage antibiotic
research and development, especially in the search for new classes of compounds, so the
return on investment in this area is low or negative. . Finding a new antibiotic candidate and
bringing it to market can take 8 to 20 years.

This is where phage therapy comes in. Phage therapy uses bacteriophage viruses to treat
bacterial infections and has been around for over a century. Bacteriophages are small viruses
that can target and kill certain types of bacteria without affecting other types of microbes in
our bodies. Commercialization in this area has been slow, due to the availability of antibiotics
now, but the practice is gaining momentum.

In a recent article, Nature Biotechnology takes a look at biotech companies involved in this
field. The phage therapy market is expected to grow 17% by 2030, reaching $84 million
annually. Big Pharma isn't on board yet, but that may change. Why is this happening now?
We've known about antibiotic-resistant bacteria for decades, and phage therapy has been
around for a long time. It seems to be a combination of things.

First, and most importantly, scientific research can explain phages-how to identify them and
what they can do. Finding the right phage to fight a bacterium takes time. To find phages that
kill specific bacteria, you need to test a library of phages with strong interactions. In the past,
scientists have used several methods to compare phages to bacteria: microfluidic PCR,
PhageFISH, and tests such as the spot test and the agar plating test. Although these methods
are very accurate, they are not fast or easy.

Computer methods can identify interactions. This includes a reference approach based on
current virus sequence databases. However, this method is less sensitive and can identify
false positive results because viral sequences are similar in number to bacterial sequences.
The new reference-free method uses machine learning algorithms to simplify this process.
This approach is only possible because of the large datasets available to train and test
machine learning algorithms and determine phage matches. Of course, computer predictions
still need to be verified, and more data is needed to move forward, but these methods can
help in the search. Scientists are also trying to engineer genes to target specific bacteria by
modifying phage receptor-binding proteins (RBPs), thereby bypassing the long-term search
for natural phages that can bind to bacteria. A high-throughput RBP differentiation strategy
has been developed to screen for mutants with extended host ranges. Synthetic biology
approaches can also improve the ability to kill bacteria by using engineered phages that
contain antibacterial genes or proteins. This is another area where machine learning will lead
to better phage designs.

Phage production also needs to be improved to ensure that this medicine is available to the
wider community. Currently, the phage therapy is still being tested and is applying on a case-
by-case basis for a one-time New Drug Approval from the US Food and Drug Administration
(FDA). To use phage therapy, the case must be dire and the disease must be multidrug-
resistant. Centers like IPATH at UC San Diego can help patients get these phage treatments,
and many clinical trials are underway.

Finally, the COVID-19 pandemic also plays a role. In a study in Dutch hospitals, secondary
bacterial infections were rare (1.2%), but more than 60% of patients received antibiotics. It is
becoming increasingly clear to the public that there are other ways to stop the spread of these
antibiotic-resistant bacteria, which are caused by the overuse of antibiotics.

Challenges remain. Currently it is not possible to produce and distribute phage drugs on a
large scale. Many phages are possible, so clinical trials of treatment dosage should be
carefully considered. It's important to understand that bacteria's job is to evolve - they've been
doing this for hundreds of millions of years, not just to respond to antibiotics. Phage
resistance can occur, although we have also seen that this can be avoided by targeting
bacteria using a combination of phages, as it is unlikely that a single strain will develop
resistance to phages. all of the mixture. Finally, phages are illegal drugs, not only in the US
but also in the UK and the EU. The FDA seems to agree, but there are no guidelines for
introducing these phages into the clinic.

Phage therapy may not replace the widespread use of antibiotics, but it is expected to prove
safe and have widespread clinical utility, making it a much-needed addition to the antibiotic
arsenal.

5.3 ETHICAL CONSIDERATIONS

Phage therapy, which involves the use of bacteriophages (viruses that infect and kill bacteria)
to treat bacterial infections, raises various ethical considerations. While the potential benefits
of phage therapy are promising, it's essential to address ethical concerns to ensure the
responsible and ethical development and application of this therapeutic approach.

Safety and Efficacy: Before implementing phage therapy, it is crucial to ensure that the
treatment is safe and effective. Ethical considerations include conducting rigorous research
and clinical trials to establish the therapy's safety profile and efficacy. Patient safety should
be a top priority, and potential risks and benefits must be thoroughly evaluated.

Regulatory Oversight: Ethical use of phage therapy involves adherence to regulatory

standards. There should be a framework in place to oversee the development, production, and
distribution of phage-based products. Regulatory bodies play a crucial role in ensuring that
these therapies meet established safety and efficacy standards.

Informed Consent: Like any medical intervention, obtaining informed consent from patients
is paramount. Patients must be fully informed about the nature of phage therapy, its potential
risks and benefits, and any alternatives available. The complexity of phage therapy, including
the dynamic nature of phage-bacteria interactions, requires transparent communication
between healthcare providers and patients.

Access and Equity: Ensuring equitable access to phage therapy is an ethical concern. Issues
of affordability, availability, and accessibility need to be addressed to prevent disparities in
access to this treatment. It is important to avoid situations where phage therapy becomes
available only to those who can afford it, creating healthcare inequalities.

Antibiotic Resistance: Phage therapy is often considered as an alternative to antibiotics,

especially in cases of antibiotic-resistant bacterial infections. However, ethical considerations
include the responsible use of phages to minimize the risk of developing phage-resistant
bacteria. A balanced approach is needed to avoid the emergence of new public health
challenges.

Environmental Impact: The potential environmental impact of phage therapy, such as the
release of genetically modified phages into the environment, raises ethical questions. Careful
consideration must be given to potential unintended consequences, and efforts should be
made to minimize any negative impact on ecosystems.

Research Ethics: Ethical concerns also extend to the research phase of phage therapy
development. Researchers must conduct their studies with integrity, ensuring transparency,
honesty, and the humane treatment of research subjects, which may include both humans and
animals.

The ethics of phage therapy encompass a range of considerations, from patient safety and
informed consent to broader societal impacts. An ethical framework that prioritizes safety,
transparency, accessibility, and environmental responsibility is essential for the responsible
development and application of phage therapy.

5.4 RECOMMENDATIONS FOR FURTHER RESEARCH

This treatment is currently very demanding in terms of: B. Lifespan of individual phages or
phage combinations, antagonism and synergism of phages and/or antibiotics,
pharmacokinetics, pharmacodynamics, pharmacogenomics and host response to phage
preparations. The first and most important step in this approach is to obtain a large, widely
available phage library and an effective method for screening the phage library to isolate
target bacteria (resistant to antibiotic treatment). This is the development of a new
mechanism. Another reason is to eliminate bacterial infections (by cell lysis) in sick patients,
preferably without nasty side effects or disruption of the natural microbiome.
Phages are generally considered safe for humans because they cannot multiply within the
cells of treated microorganisms and are excreted by the patient's kidneys once the antibiotic-
resistant pathogen is killed. However, phage lysates contain many types of harmful, health-
and life-threatening products, such as endotoxins (LPS), mainly derived from Gram-negative
bacteria, and protein toxins produced by pathogenic bacterial strains. There is a possibility.
Therefore, there is a continuing need to improve methods for isolating, enriching, and
purifying phage preparations into highly potent and safe forms useful for human and animal
therapy.

Advances in technology (genetic engineering, synthetic biology, throughput sequencing,

metagenomics, etc.) are expected to make phage products available in specialized Good
Manufacturing Practice (GMP) facilities. Although it is very easy to isolate natural phages
from many pathogens, there are still many bacterial species for which phages are rarely or
unavailable. This means that new sources for obtaining and isolating phages across a broad
host range need to be identified.

PT may represent a new and widespread approach to combating resistant pathogens in the
future. Therefore, logistical and regulatory hurdles must be overcome to make PT a widely
available alternative treatment for patients who have failed antibiotic therapy. Moreover,
further clinical studies are still needed to supplement clinical data on phage biology, phage
preparation dosage, and PT administration methods.
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