The phage therapy as a promising treatment method

following search criteria were applied: (1) retrospective studies of phage
therapy against pseudomonas aeruginosa
that cause infection to the respiratory tract, ear and burn wounds (2) full-text
research articles with in-vivo studies on either pre-clinical animal models or
clinical human models (3) studies considering the effectiveness of phage
therapy by using colonies-forming units (CFU) or phage-forming units (PFU)
counts or animal models survival rate as outcome criteria and (4) studies in
English. Since the use of phage as therapeutic agent has been proposed in 1917
and many studies have been performed after, this systematic review search
comprise of the time period between January 2007 and December 2017.

Selection Criteria

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publications on the efficacy of phage therapy against pseudomonas aeruginosa were identified by searching databases such
as PudMed, Aston Library and ScienceDirect as well as additional sources from
bibliographies of several articles. The date of search was 9 December 2017, 19
December 2017 and 25 December 2017 respectively. “Bacteriophage” and “pseudomonas aeruginosa” served as

Search Method


Figure 1. Results of
systematic literature search

Materials and Method



one of the leading opportunistic pathogens involved in nosocomial infection, P. aeruginosa has been the focus of
phage application and genotyping studies. Recent studies of both in-vitro and
in-vivo, as well as in animals and human models have shown phage therapy as a
promising treatment method against P.
(Wrigh et al., 2009; Hagens
et al., 2006; Rhoads et al., 2009; Hurley et al., 2012) To date, there are a total of 137 phages known to target
Pseudomonas species. Of that, 94.2%
of the phage belongs to the Caudovirales
order that comprises of phages with short and noncontractile tail (Podoviridae); phages with a
long and contractile tail (Myoviridae),
and phages with a long and noncontractile tail (Siphoviridae).
Among the Pseudomonas genus phages
that are sequenced, approximately 60% of them are lytic phages and holds
potential therapeutic function against Pseudomonas
species. (Ackermann, 2009; Sillankorva and Pires, 2014; Ceyssens and Lavigne, 2010)

Bacteriophages were discovered almost a century ago. (Pires et al., 2015) It was
proposed by Félix
d’Hérelle, a French-Canadian microbiologist in 1917, regarding the use of phage as antimicrobial agents. (Pires et al., 2015) Following D’Hérelle’s finding, phage therapy was used to
treat dysentery in 1919, staphylococcal skin disease in 1921 as well as several
studies as reported by Thurman B. R., Schless R. A.  and Stout B. F. (Sulakvelidze
et al., 2001) Therapeutic phages were also actively produced by
commercial laboratory against various bacterial infections including
suppurating wounds and upper respiratory tract infections. (Pires et al., 2015) However, with the introduction of antibiotics, phage
therapy has been superseded in most of the Western world but continue to
be used in Eastern Europe and the former Soviet Republic of Georgia.  (Sulakvelidze
et al., 2001) Antibiotics were seen as the choice of treatment against
bacterial infection as they are cheap and effective. Yet ironically, the use of
antibiotics promotes the emergence of anti-microbial resistance bacteria. (Pires et al., 2015) Moreover, with several distinctive advantages of
bacteriophage over antibiotic therapy such as bacterial specificity and
effectiveness against biofilm, a renew interest in phage therapy has arise. (Azeredo
and Sutherland, 2008) 

Phage therapy involved the use of bacteriophages, in
replacement of antibiotics, to treat bacterial infection. (Abedon,
2015) It is one of the potential therapeutic approaches in view to the
emergence of multidrug resistance bacteria. (Abedon
et al., 2011) Bacteriophages, or phages,
are viruses that specifically infect and kill bacteria. They are divided into
two classes based on their life cycle, namely the lytic phage and temperate
phage. (Guttman et al., 2004) Lytic phage infects bacteria
by injecting and replicating its genome into the bacterium, followed by
inducing host cell lysis. This results in the release of new viral particles to
start another round of infection, for as long as sufficient bacteria host is
present. (Harper and Kutter, 2008)  On the other hand, temperate phage, which
undergo the lysogenic cycle, infects bacteria by injecting and integrating its
genome into the host chromosome. The integrated viral genome, or prophage,
replicate with the host cell DNA and unless activated by specific stimuli, will
remain latent in the daughter cells. (Harper
and Enright, 2011; Little, 2005) For therapeutic purposes, only bacteriophages that are obligate
lytic in nature are desired due to their rapid killing of host cell. (Harper and Enright, 2011)

aeruginosa also has an ability to produce biofilm, which are slimy
extracellular polymeric substance (EPS) matrix,. (Taylor et
al., 2014)
These biofilm have a 10-1000 fold higher resistance against
antimicrobial killing comparing to planktonic bacteria cells. (Hoyle and Costerton, 1991) Biofilms are aggregate of cells that first
adherent irreversibly to a surface through the action of pili and flagella.  EPS matrix is then produced by the cells to
promote stronger cell adherence and confers biofilm structure. (O’Toole
and Kolter, 1998; Toutain et al., 2007) This matrix, though allows
nutrients and small molecules to penetrate, act as a barrier for effective
antimicrobial activity and host immune defence. (Olson et al., 2002) It
is proposed that this gain of resistant mechanism in P. aeruginosa is the result of adaptive ability by modifying gene
expression and external environment conditions. The high resilient to physical
stress and antibiotic clearance has made P.
aeruginosa a major therapeutic concern within healthcare setting,(Taylor et
al., 2014) P. aeruginosa biofilm commonly colonized
medical devices and epithelial cells such as ventilators and open wounds,
resulting in infections that are difficult to eradicate using conventional
therapeutic methods, (Bjarnsholt et al., 2009; Boucher et al., 1997, Worlitzsch
etal., 2002) An example is the colonization of P. aeruginosa in CF lungs. The formation of biofilm within CF lungs
results in chronic infection that, despite all means of antibiotic therapies,
persists for life, (Taylor et al., 2014)  Hence, the development of new
antimicrobial agents and strategies are at a growing demand against these
pathogenic bacteria.

treatment against P. aeruginosa
infection involved the use of anti-pseudomonal agents such as ?-lactams, aminoglycosides, fluoroquinolones
and more recently, polymyxins.  (Mesaros
et al., 2007).However, the high
intrinsic, acquired and adaptive resistance ability displayed by P. aeruginosa makes its eradication a
challenge. (Taylor et al., 2014)  Intrinsic resistance refers to the
innate ability of wild type bacteria to limit its susceptibility to
antibiotics. P. aeruginosa possess
this underlying ability due to the low permeability of its outer member. (Giamarellou,
2002) As the outer membrane of P aeruginosa is 12-100 fold less permeable than other gram negative
bacteria, (Hancock and. Bell, 1998) it serves as a barrier slowing down
antibiotic penetration through the water-filled porin channels. Also, the
expression of efflux pump and production of enzymes that inactivate antibiotic
synergizes with the slow antibiotics uptake, making P. aeruginosa Intrinsically more resistance. (Giamarellou,
2002) The Multidrug
efflux systems such as the MexAB-OprM, MexEF-OprN and MexCD-OprJ confer
resistance to ?-lactam, fluoroquinolones and aminoglycoside (Chatterjee
et al., 2016) while inactivating enzymes such as AmpC ?-lactamase
degrade slow flowing antibiotic that are penetrating across the outer membrane.
et al., 2014) Acquired resistance is the consequence of antibiotic
exposure resulting in mutation of chromosomal gene or acquiring of antibiotic
resistance gene. (Pires et al., 2015)
P. aeruginosa has one of the
largest genome within the prokaryotic world that encodes for large amount of
proteins responsible for its pathogenesis. Intrinsically, 0.3% of its coding
gene possesses antimicrobial function, (Mesaros et al., 2007) With
5570 open reading frames, the large genome is also highly flexible in acquiring
mobile genetic elements such as plasmids and integrons that confer antibiotic
ability. (Mesaros et al., 2007; Taylor et al., 2014).  Adaptive resistance, on the other hand,
is dependent on the growth conditions and environmental stimuli to trigger
cellular regulatory events and this susceptibility are usually revert with the
removal of these triggers, (Fernández et al., 2011) Conditions such
as antibiotic exposure, DNA stress, heat shock and environmental PH has been
reported to induce adaptive resistance. (Fernández et al., 2011) The ability of P. aeruginosa to thrive in diverse
environment and resist a range of antibiotics is probably due to its large and
complex genome as adaptive resistance involved many genes including resistomes,
explaining the high
versatility and adaptive capacity of the species, (Taylor et
al., 2014)

of P. aeruginosa are difficult to
treat as the bacteria possess an arsenal of virulence factor that helps in its
pathogenesis and invasion of host cells. The extracellular virulence factors
include proteases which degrades and inhibits complement proteins activation (Chatterjee et al., 2016), Elastase (LasA and LasB),  (Kessler et al., 1997; Grande et
al., 2007) exotoxin A which inhibits protein synthesis in eukaryotes, (Stuart
and Pollack, 1982) phospholipases that causes erythrocytes hemolysis, (Shortridge
et al., 1992) exoenzymes that promote invasion and chronic infections by
disrupting cytoskeletal structure (Sadiko et al., 2005) while
the cell-associated factor includes lipopolysaccharide (LPS), flagella, and
pili that facilitate P. aeruginosa colonization, survival and invasion within
host tissues. (Chemani et al., 2009)

P. aeruginosa is capable of causing
infections that are both acute and chronic. One such example is infections to
the respiratory tract. The environment of the respiratory tract allows P. aeruginosa to adapt and grow well,
resulting in potential pneumonia or lung infection, especially in those who are
immunocompromised or hospitalised within intensive care units. (Mesaros
et al., 2007) Hospital-acquired pneumonia is the second leading complication
of P. aeruginosa infection. (Torres
et al., 2010) It is usually ventilator-associated where pneumonia
develops after more than 48 hours of mechanical ventilation or intubation. (Bielen
et al., 2017) Another
susceptible group are individuals with cystic fibrosis (CF). CF
is a heterogeneous recessive genetic disorder due to mutation of the CF transmenbrance
regulator (CFTR) gene. (Knowles and Durie, 2002) This
gene helps in the regulation of transporting chlorine ions across the
epithelia, necessary for the production of mucus that lubricates the airway. In
classic CF, a loss of function due to mutation to both the CFTR gene leads to airway
dehydration and an impaired mucociliary clearance of bacteria, allowing
bacteria to colonize the lungs more readily. (Taylor et al., 2014) P. aeruginosa is also the predominant agents causing acute diffuse
otitis externa, commonly known as swimmer’s ear. (Mesaros et al., 2007) It
is characterized by erythema, otalgia and itch as early symptoms and may
progress to having aural fullness, discharge and conductive hearing loss. (Nuttall,
2016) This type of otitis externa is known as
swimmer’s ear as swimmers are at a risk that is 5 times greater than
non-swimmers while cases of otalgia are 2.4 times greater in swimmers than
non-swimmers. (Hoadley and Knight, 1975) Besides the heat
and humidity that cause swelling of the skin, moisture from swimming increases
skin maceration of the external auditory canal. This encourages breaching of
the protective skin barrier, allowing viable pathogenic organism in the marine
waters, such as P. aeruginosa, to
emerge and become infective. (Wang et al., 2005) Burn wounds are
highly susceptible to infections especially by P. aeruginosa where complications such as bacteraemia and septicaemia are often
serious and life-threatening. (Mesaros et al., 2007) Other
than maintain body fluid homeostasis and thermoregulation, an intact human skin
also served as a physical barrier against microbial invasion. Following severe
thermal injury, extensive skin surface of the affected area are breached. (Elsayed
et al., 2006) Since P. aeruginosa
is so commonly found in the environment, burned individual are likely to be
colonized with this microorganisms before the burn wounds are heal completely. (Lyczak
et al., 2000)

Pseudomonas aeruginosa is a gram
negative bacillus that acts as an opportunistic human pathogen. It is
considered as one of the nosocomial causative agents and is responsible for the large scale multi-drug resistant
infections. (Pier and Ramphal, 2005) Rarely,
P. aeruginosa infects healthy
individual. However, it is capable of infecting all tissue when the physical
barrier is breached or in individuals whose immune defence is compromised such
as those with cystic fibrosis and cancer. (Morrison and Wenzel, 1984)
Hence, this explains why P. aeruginosa
is a major concern within healthcare settings and their infections are often
severe and life-threatening. (Maschmeyer and Braveny, 2000) Each
year, it is estimated that there are 51,000 cases of hospital acquired P.
infections within the United States. Of that, multidrug-resistant P. aeruginosa strains accounts for
around 13% of the cases, contributing to roughly 400 deaths annually. (Chatterjee et al., 2016)

The increased incidence of antimicrobial resistance has been
one of the pressing problems faced by healthcare services worldwide. Bacteria
are gaining resistance to most of the currently available
antimicrobial agents which results in significant increased morbidity, mortality and healthcare costs. (Sulakvelidze
et al., 2001) It is estimated that these
infections affects 2.5 million people annually and has claimed the lives of at
least 50 000 people across Europe and USA alone. (Centers for Disease
Control and Prevention, 2013) The emergence of
multidrug-resistance bacteria has also raised the concern for nosocomial
infections. It is define as infections acquired during hospital stay and was
absence at the point of admission, (Ducel
et al., 2002) as well as potential occupational
infections among staff within the facility. (Benenson, 1995) An
incidence survey conducted by the World Health Organization (WHO) across 14
countries within Europe, Eastern Mediterranean, South-East Asia and Western
Pacific that involved 55 hospitals has shown that an average of 8.7% of
hospitalized patients acquire nosocomial infection. Thus,  this means that at least 1.4 million people
worldwide are suffering from complications acquired in hospital at any point of
time. (Tikhomirov, 1987)