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Alexandria Journal of Medicine
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/tajm20
A review of phage mediated antibacterial
applications
Kenneth Ssekatawa , Denis K. Byarugaba , Charles D. Kato , Eddie M.
Wampande , Francis Ejobi , Robert Tweyongyere & Jesca L. Nakavuma
To cite this article: Kenneth Ssekatawa , Denis K. Byarugaba , Charles D. Kato , Eddie M.
Wampande , Francis Ejobi , Robert Tweyongyere & Jesca L. Nakavuma (2021) A review of
phage mediated antibacterial applications, Alexandria Journal of Medicine, 57:1, 1-20, DOI:
10.1080/20905068.2020.1851441
To link to this article: https://doi.org/10.1080/20905068.2020.1851441
© 2020 The Author(s). Published by Informa
UK Limited, trading as Taylor & Francis
Group.
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Published online: 31 Dec 2020.
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REVIEW ARTICLE
A review of phage mediated antibacterial applications
Kenneth Ssekatawa
a,b,c
, Denis K. Byarugaba
a
, Charles D. Kato
a
, Eddie M. Wampande
a
, Francis Ejobi
a
,
Robert Tweyongyere
a
and Jesca L. Nakavuma
a
a
College of Veterinary Medicine Animal Resources and Biosecurity, Makerere University, Kampala, Uganda;
b
Department of Biochemistry,
Faculty of Biomedical Sciences, Kampala International University-Western Campus, Bushenyi.;
c
African Center of Excellence in Materials
Product Development and Nanotechnology (MAPRONANO ACE), College of Engineering Design Art and Technology, Makerere University,
Kampala, Uganda
ABSTRACT
Background: For over a decade, resistance to newly synthesized antibiotics has been observed
worldwide. The challenge of antibiotic resistance has led to several pharmaceutical companies
to abandon the synthesis of new drugs in fear of bacteria developing resistance in a short
period hence limiting initial investment return. To this eect, alternative approaches such as
the use of bacteriophages to treat bacterial infections are being explored. This review explores
the recent advances in phage-mediated antibacterial applications and their limitations.
Methods: We conducted a comprehensive literature search of PubMed, Lib Hub and Google
Scholar databases from January 2019 to November 2019. The search key words used were the
application of bacteriophages to inhibit bacterial growth and human phage therapy to extract
full-text research articles and proceedings from International Conferences published only in
English.
Results: The search generated 709 articles of which 95 full-text research articles fullled the
inclusion guidelines. Transmission Electron Microscopy morphological characterization con-
ducted in 23 studies registered Myoviruses, Siphoviruses, Podoviruses, and Cytoviruses phage
families while molecular characterization revealed that some phages were not safe to use as
they harbored undesirable genes. All in vivo phage therapy studies in humans and model
animals against multidrug-resistant (MDR) bacterial infection provided 100% protection. Ex vivo
and in vitro phage therapy experiments exhibited overwhelming results as they registered high
ecacies of up to 100% against MDR clinical isolates. Phage-mediated bio-preservation of
foods and beverages and bio-sanitization of surfaces were highly successful with bacterial
growth suppression of up to 100%. Phage endolysins revealed ecacies statistically compar-
able to those of phages and restored normal ethanol production by completely eradicating
lactic acid bacteria in ethanol fermenters. Furthermore, the average multiplicity of infection
was highest in ex vivo phage therapy (557,291.8) followed by in vivo (155,612.4) and in vitro
(434.5).
ARTICLE HISTORY
Received 19 August 2020
Revised 4 November 2020
Accepted 10 November 2020
KEYWORDS
Antibiotics resistance;
bacteriophages; phage
therapy; phage mediated
biocontrol; phage efficacy
1. Background
Currently, the world populace is deemed to be at
a great risk as a result of the ever-escalating prevalence
of antibiotic resistance bringing about an epoch where
many familiar bacterial infections are becoming
increasingly hard to treat [1]. Similar to many other
developing countries, Sub-Saharan Africa is experien-
cing an elevated burden of bacterial infectious diseases
which calls for the overuse of antibiotics and conse-
quently emergence of resistant microorganisms [1,2].
The development of antibiotic resistance is also con-
tributed by self-medication with uncontrolled over-
the-counter access to drugs without any guidance
from qualified medical practitioners. In addition,
there is excessive application of antibiotics in poultry,
aquaculture, and livestock production. The unrest-
ricted access and use of antibiotics for animal disease
treatment and prophylaxis as well as growth promo-
tion have been implicated as one of the major drivers
for antibiotic resistance that may spillover to humans
[3–5]. Infectious food and water-borne illnesses are
acquired through the consumption of contaminated
food and water; and are the major cause of mortality
and morbidity worldwide owing to their extensive and
spontaneous transmission [6,7]. It was estimated that
water, sanitation, and hygiene (WSH) associated
infectious diseases are accountable for 4.0% of the
worldwide deaths and 5.7% of the universal disease
burden [7,8]. Furthermore, WHO reported that
600 million or 1 in 10 people fall ill worldwide as
a result of foodborne infections and more than
91 million people affected are in Africa [6].
The rate at which drug resistance emerges has
resulted in big pharmaceutical companies backing
away from developing new antibiotics since the latter
CONTACT Dr. Jesca L. Nakavuma jesca.nakavuma@gmail.com College of Veterinary Medicine, Animal Resources and Biosecurity, Makerere
University, Kampala, Uganda
Supplemental data for this article can be accessed here.
ALEXANDRIA JOURNAL OF MEDICINE
2021, VOL. 57, NO. 1, 1–20
https://doi.org/10.1080/20905068.2020.1851441
© 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits
unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
are rendered non-effective within a short period, mak-
ing the venture not cost-effective [9]. Therefore,
affordable alternative approaches such as the use of
probiotics, phytomedicines, and bacteriophages to
manage bacterial infections and control the emergence
of antibiotic resistance are highly commendable.
Bacteriophages (phages) are natural enemies of bac-
teria which are the most abundant replicating entities
on earth. Phages are viruses that specifically attack and
multiply in bacterial cells and have no effect on other
cell types. They are self-replicating and self-limiting as
long as the specific bacterial host cells exist. Similar to
other viruses, their genomes may either be double-
stranded or single-stranded DNA or RNA [10].
Phages have either a lytic or lysogenic type of replica-
tion cycle. The lytic cycle, also referred to as the virulent
cycle, results in the production of progeny viruses that
are released through cell lysis. The lysogenic or tempe-
rate cycle results in the incorporation of the phage
genome into the host chromosome without the produc-
tion of new virus particles. Depending on some circum-
stances, some phages can exhibit both replication cycles
[10]. Lytic phages are applied as bacterial growth inhi-
bitors, which can be categorized as phage therapy or
phage-mediated decontaminants. For therapy, phages
are mainly used like antibiotics, whereas for deconta-
mination, they are applied as disinfectants. Literally,
phage therapy is the application of phages as therapeu-
tic agents more especially in a clinical context to treat
bacterial infections while phage-mediated biocontrol
can be defined as the use of phages to suppress bacterial
growth on non-living surfaces. Safety and efficacy of
phage therapy or phage-mediated biocontrol relies on
isolation and use of only professional lytic phages,
which are obligately lytic or virulent but they are
neither temperate nor directly linked to temperate
phages [11]. Phage therapy is a proven eco-friendly
alternative approach to prevent and control pathogenic
bacterial infections [12,13].
Phages were used to treat bacterial infections in
Europe during the pre-antibiotic era. However, with
the discovery of antibiotics and the substandard med-
ical trials conducted in the western world without
putting into consideration that phages were specific,
phage therapy was shortly after deemed impotent in
the treatment of bacterial infections. Nevertheless,
phage therapy continued to be used for the treatment
of bacterial infections in the Soviet Union since 1940
[14]. The advantages of phage applications, such as
disruption of bacterial biofilms and nondependency
on the drug resistance status of the organisms, have
rekindled their use as antibacterial agents [15,16].
Furthermore, renewed attention to phage therapy has
been registered due to an overall decline in the total
reserves of effective antibiotics. Hence, phage therapy
clinical trials and experiments in poultry, aquaculture,
crop husbandry, model animals, in vitro model
systems, and humans have been widely carried out
[17,18]. Currently, the notable human phage therapy
under application is the compassionate use of phages
as individualized therapeutic options to manage MDR
bacterial infections unresponsive to all classes of con-
ventional antibiotics [19]. Furthermore, phage pre-
parations have been used and experimented with as
diagnostic tools for bacterial infections to supplement
the available methods [12].
For use as decontaminants, several studies have been
conducted to evaluate the efficacy of phages as bio-
control agents against food and beverage borne patho-
gens [20]. Phages have been experimented with in bio-
sanitization of equipment surfaces to eradicate biofilms
in food industries [21]; and bio-preservation of perish-
able processed foods to increase shelf-life. Some phage-
specific enzymes; such as lysins which degrade the cell
wall of gram-positive bacteria, have been applied to
processed foods to enhance their safety for human
consumption [18,22–24]. The use of bacteriophages in
food products in the US, Europe, and Australia has been
reported [25]. Indeed, some phage preparations have
been approved in the USA and are commercially avail-
able; such as LISTEX P100; LMP-102
TM
, Listshield
TM
,
ECP-100
TM
(Ecoshield
TM
), SALMONELEX
TM
,
AgriPhage
TM
, and Biophage-PA [26].
This review expounds on the current level, limita-
tions, and prospects of phage applications such as
enhancing food safety and fermentation of biofuels;
phage therapy clinical trials and experiments in
humans and model animals; animal and plant disease
control and environmental bioremediation.
2. Methods
2.1. Literature search strategy
A comprehensive literature search of PubMed, Lib
Hub, and Google Scholar databases was conducted
from January 2018 to November 2018. The search
key words used were “application of bacteriophages
to inhibit bacterial growth” and “human phage ther-
apy,” Figure 1.
3. Study selection criteria
The search targeted articles published in English with-
out restriction on year of publication in an attempt to
capture all available literature about the application of
phages as antibacterial agents worldwide, Figure 1. In
addition, only full-text research articles and proceed-
ings from the International Conference on Prevention
& Infection Control were selected, Table 1–5, S1.
Review articles were excluded from this search. To
avoid bias, all the seven coauthors were involved in
the selection process. Articles were assigned to the
different coauthors blindly, review reports on the
2K. SSEKATAWA ET AL.
merits and demerits of the studies as per inclusion
criteria were submitted to the lead researcher (JLN)
and the entire selection process was conducted based
on the review reports by all the seven coauthors. In
case of any disagreement, powers were entrusted to
the most experienced researchers in bacteriophages
(JLN, DKB, and FE) to make the final decision.
4. Data extraction
A database was created in which the field of phage
application, type of phage or phage part used, source
of phages, level of phage application, type of bacteria
and strain or serovar challenged, level of phage effi-
cacy, physiochemical properties of phages, the multi-
plicity of infection (MOI) of phages and methods
used in the characterization of phages were included.
Studies where MOIs were not reported but the num-
ber of plaque-forming units/mL (PFU/mL) and the
number of colony-forming units/mL (CFU/mL)
given, MOIs were computed by dividing the PFU/
mL by CFU/mL units (O’Flynn et al., 2004). To
compare the MOI of different investigations, all stu-
dies were grouped into three categories namely;
in vivo phage therapy, ex vivo phage therapy, and
in vitro phage therapy.
Figure 1. Selection process of research articles for inclusion in this review.
Table 1. In vivo human phage therapy trials.
Phage therapy in humans Phage type Source of phages Pathogens targeted
Serovar/
pathotype efficacy Ref
Treatment of diabetic toe
ulcers
Staphylococcal phage Sb-1 Eliava Institute S. aureus (MRSA and
MSSA)
- 100% [93]
Treatment of GIT MRSA
infection
polyvalent S. aureus bacteriophages L. Hirszfeld Institute
collection
S. aureus (MRSA) - 100% [94]
Treatment of burn infections - J. Soothill P. aeruginosa - 100% [95]
Treatment of infected
venous stasis ulcers and
other poorly healing
wounds
Pyophage in PhagoBioDerm films Eliava Institute P. aeruginosa, E. coli,
S. aureus, Proteus,
and Streptococcus
- 76% [49]
Treatment of corneal
abscess and interstitial
keratitis
S. aureus bacteriophage SATA-8505 ATCC VRSA - 100% [96]
Treatment of
burn wound infection
Cocktail of P. aeruginosa phages 14/1
(Myoviridae) and PNM (Podoviridae)
and S. aureus phage ISP (Myoviridae)
Merabishvili et al 2009 S. aureus and
P. aeruginosa
- 0% [69]
Treatment of chronic otitis
antibiotic-resistant
P. aeruginosa
Infection
Biophage-PA NCIMB MDR P. aeruginosa - 80% [97]
Treatment of P. aeruginosa
UTI
PA Phage cocktail (Pyophage #051007) Eliava Institute MDR P. aeruginosa - 100% [98]
Treatment of acute bacterial
diarrhea
T4-like coliphages cocktail Microgen-Russia E. coli - 0% [70]
Treatment chronic bacterial
prostatitis
IIET bacteriophage collection IIET bacteriophage
collection sewage,
environmental, or
drinking water
Enterococcus faecalis - 100% [99]
Phage safety analysis Phage cocktail Coli Proteus Microgen Russia E. coli and proteus - - [30]
ALEXANDRIA JOURNAL OF MEDICINE 3
Table 2. Phage-mediated biocontrol of bacterial growth in ethanol fermentation, foods, and beverages.
Field of application Phage/phage part used Source of phages/part
Level of
application Bacteria type controlled Bacteria serotype/Strain Efficacy Ref.
Ethanol fermentation Streptococcal phage LambdaSa2 (λSa2) endolysin EMD Biosciences, San
Diego,CA)
Laboratory
experiment
Lactobacillus, staphylococci, and
streptococci
77.3% [100]
LysA, LysA2, LysgaY and λSa2 endolysin proteins Subcloned into the pET21a
E. coli expression vector
Ethanol fermentation Lytic enzymes LysA and LysA2 endolysins genes expressed
in Saccharomyces
cerevisiae
Laboratory
experiment
Lactobacillus fermentum,
Lactobacillus brevis, and
Lactobacillus mucosae.
~ 90% [101]
Ethanol fermentation EcoSau and EcoInf Wastewater influent Laboratory
experiment
L. fermentum 100% [37]
Ethanol fermentation ATCC® 8014-B1™ (phage B1) and ATCC® 8014-B2™ (phage
B2)
ATCC Laboratory
experiment
Lactobacillus plantarum ATCC®
8014™
ATCC® 8014™ 99% [92]
Dairy (Cheese) Phage P100 Dairy plant sewage effluent Laboratory
experiment
L. monocytogenes WSLC 1001 100% [39]
Dairy (Milk) vB_SauS-phiIPLA35 Dairy environment Laboratory
experiment
Staphylococcus aureus 100% for
cocktail [102]
Dairy (Milk) and control of E. coli
biofilms
BECP2 and BECP6 phages Sewage Laboratory
experiment
E. coli O157:H7 90% [103]
Dairy (Milk fermentation) Coliphages DT1 and DT6 Feces Laboratory
experiment
E. coli O157:H7 STEC 100% [104]
Dairy (Cheese) phage A511 - Laboratory
experiment
Listeria monocytogenes, 90% [105]
Fruits (Cucumber, Apple, and
Tomatoes)
T7 bacteriophages Laboratory
experiment
Escherichia coli BL21 99.9% [106]
Fresh-cut fruits and vegetables LM-103 and LMP-102, Intralytix, Inc. (Baltimore,
Md.).
Laboratory
experiment
Listeria monocytogenes, 99.9% [107]
Dairy, poultry, beef products, sea
food, and vegetables
A511 and P100 - Laboratory
experiment
Listeria monocytogenes WSLC 1001 (serovar 1/2 c)
and Scott A (serovar 4b)
100% [108]
Beer industry Myophage SA-C12 Fresh silage Laboratory
experiment
Lactobacillus brevis 8840 (NCIMB culture
collection),
100% [109]
Chicken cuts FSP-1 and FSP-3/PSZ1 and/PSZ2 Laboratory
Experiment
Salmonella enterica Strain S49 92% [110]
Spinach - Feedlot cattle feces Laboratory
Experiment
Escherichia coli O157:H7 100% [111]
Oysters Siphoviridae phage pVp-1, - Laboratory
experiment/trial
on oysters
Vibrio parahaemolyticus CRS 09–17 ~99.999% [112]
Fermented Soy bean paste BCP1-1 and BCP8-2 Fermented food products Laboratory
experiment
Bacillus cereus ATCC27348, ATCC21768,
ATCC13061
100% [113]
Bioactive packaging materials
(meat and alfalfa seeds and
sprouts)
LinM-AG8, LmoM-AG13, and LmoM-AG20, while the E. coli
O104:H4 EcoM-HG2, EcoM-HG7 and EcoM-HG8
(Myoviridae)
Canadian Research Institute
for Food Safety
Laboratory
experiments
Listeria monocytogenes and
Escherichia coli
E. coli O104:H4, LJH391
serotype 1/2b,
100% [114]
Infant formula milk ESP 1–3 and ESP 732–1 Sewage Laboratory
experiment
Enterobacter sakazakii ATCC 29,544, 236/04, 732/03 100% [115]
Infant formula milk leB, leE and leN Slurry Laboratory
experiment
Cronobacter sakazakii C. sakazakii ATCC BAA 894,
C. sakazakii ATCC BAA 894
LUX
100% [40]
(Continued)
4K. SSEKATAWA ET AL.
Table 2. (Continued).
Field of application Phage/phage part used Source of phages/part
Level of
application Bacteria type controlled Bacteria serotype/Strain Efficacy Ref.
Pork, milk, and kitchenware fHe-Yen3-01 (Podoviridae) fHe-Yen9-01, fHe-Yen9-02, fHe-
Yen9-03 (Myoviridae)
Sewage Laboratory
experiment
Yersinia enterocolitica O:3 strain 6471/76 and O:9
strain Ruokola/71
100% [41]
Milk, sausage, and lettuce LPST10, LPST18, and LPST23(Siphoviridae family) Waste water, sewage, farm
ditch, poultry house
Laboratory
experiment
Salmonella strains Typhimurium and
Salmonella Enteritidis
Salmonella Typhimurium
ATCC 14,028
64.1% [116]
Active food packaging system
(cellulose acetate films)
BFSE16, BFSE18, PaDTA1, PaDTA9,PaDTA10 and PaDTA11 Chickenfeces, poultry
exudates, and swine
feces
Laboratory
experiment
Salmonella enterica subsp. Enterica serovar
Typhimurium ATCC 14,028
100% [117]
Bioctive food packaging system BFSE16, BFSE18, PaDTA1, PaDTA9, PaDTA10 and PaDTA11 Poultry exudates and swine
feces
Laboratory
experiment
Salmonella enterica subsp. Enterica serovar
Typhimurium ATCC 14,028.
~ 99.99% [118]
Sea food (Cockles) phT4A, ECA2 Sewage Laboratory
experiment
Escherichia coli ATCC 13,706), 90% [119]
ALEXANDRIA JOURNAL OF MEDICINE 5
Table 3. In vitro phage therapy against clinical isolates assays.
Field of application Phage/phage part used Source of phages/part Level of application Bacterial targeted Strain/serovar/pathotype Level of efficacy Ref.
Phage activity against STEC and EHEC clinical
isolates
CA911, CA933P, MFA933P and
MFA45D
Minced meat, pork sauasage &
bovine feces.
Laboratory experiment Escherichia coli STEC and EHEC 100% [44]
Phage-Antibiotics synergism against E. coli
biofilm
T4 bacteriophage ATTC 11,303-B4 LGC Standards, Middlesex, UK) Laboratory experiment E. coli biofilms E. coli 11,303 100% [120]
Phage activity against Bacillus pumilus Phage FBa1, FBa2, and FBa3 River water Laboratory experiment Bacillus pumilus 100% [121]
Phage activity against Salmonella Typhimurium phSE-1, phSE-2, and phSE-5 (family
Siphoviridae)
Sewage Laboratory experiment Salmonella
Typhimurium
Enterica serovar Typhimurium 99% [42]
Phage activity against S. aureus clinical isolates Sb-1 - Laboratory experiment Staphylococcus aureus - 100% [122]
Phage activity against Pseudomonas fluorescens
biofilms
Phage IBB-PF7A sewage treatment plant Laboratory experiment P. fluorescens biofilms 91% [123]
Phage activity against SalmonellaTyphimurium P22-B1, P22, PBST10, PBST13, PBST32,
and PBST 35)
ATCC and Hankuk University of
Foreign Studies
Laboratory experiment SalmonellaTyphimurium KCCM 40,253, ATCC 19,585,
ATCC 19,585, and CCARM
8009.
54% [124]
Phage activity against Staphylococcus aureus
biofilm
Phage DRA88 and SAB4328-A Sewage Laboratory experiment
and ex vivo-burn
models
Staphylococcus aureus
biofilm
RN6390B, RN6911, B4328,
MSSA 3, MSSA 10, MRSA 82
Reduced
biofilm
formation
[125]
Phage activity against Pseudomonas aeruginosa
biofilm
DL 52, DL 54, DL 60, DL 62, DL 64, and
DL 68
Crude sewage Laboratory experiment P. aeruginosa biofilm PAO1, PA45311, PA45291,
PA45235
95% [35]
Phage activity against Staphylococcus aureus
biofilm
DRA88 and phage K Crude sewage Laboratory experiment Staphylococcus aureus
biofilm
1598, MRSA 252, & H325 100% [36]
Phage therapy against Staphylococcus aureus phage K and phage 92 ATCC Laboratory experiment MRSA/MSSA MRSA (N315, COL, Mu50)
ATCC 6538
100% [126]
Ex vivo Phage activity against catheter MRSA
biofilm
phage K ATCC Laboratory experiment
(Catheter)
MRSA biofilms Staphylococcus aureus 46,106 99% [127]
Ex vivo Phage activity against catheter Proteus
mirabilis and Escherichia coli biofilms
Escherichia coli T4 phage ATCC
11,303-B4
Bacteriofag coli-proteic, Microgen
Pharma, Russia
Laboratory experiment
(Catheter)
Proteus mirabilis and
E. coli biofilms
E. coli ATCC 11,303 and
P. mirabilis 13 HER1094
99.99% [128]
Phage activity against Pseudomonas aeruginosa
biofilm
- Hospital environmental dirt, sewage
disposal, and cattle waste
effluents
Laboratory experiment P. aeruginosa biofilms - 50% [129]
Phage therapy against Staphylococcus aureus P128 proteins Inducible T7 expression system in
E. coli ER2566 strain
Laboratory experiment Staphylococcus aureus BK#13,725, BK#9894,
BK#13,993
99.99% [130]
Phage activity against K. pneumoniae biofilms KPO1K2 and NDP, depolymerase, and
nondepolymerase producing
phages
- Laboratory experiment K. pneumoniae biofilms B5055 (O1:K2) Significant
eradication
[131]
phage activity against MRSA & MSSA . Hospital environmental dirt, sewage
disposal, and cattle waste
Laboratory experiment MRSA and MSSA 100% for MSSA
and 78%
MRSA
[132]
Phage activity against MDR Acinetobacter
baumanni
Phage AB2 Sewage Laboratory experiment MDR Acinetobacter
baumanni
A. baumannii M3237 99.90% [56]
Phage activity against MDR P. aeruginosa Sewage Laboratory Experiment MDR P. Aeruginosa - 100% [133]
Phage therapy assay against Pseudomonas
aeruginosa and Staphylococcus spp clinical
isolates
Intesti and Pyobacteriophag Eliava BioPreparations, Tbilisi,
Georgia
Laboratory experiment P. aeruginosa and
Staphylococcus spp.
- 100% [134]
Phage bacterial lytic activity against resistant
S. aureus
SA11 (Siphoviridae family Hankuk University of Foreign studies Laboratory experiment Resistant S. aureus ATCC 13,301 and CCARM 3080 99.99& [135]
Phage bacterial lytic activity against S. aureus
biofilms isolated from orthopedic Implant
StaPhage (Myoviridae) AusPhage Pty Ltd and sewage water Laboratory experiment Staphylococcus aureus
biofilms
ORI16C02N and ORI16025 98% [136]
Phage activity against P. aeruginosa biofilms P. aeruginosa phage M4 Health Protection Agency, Colindale,
United Kingdom
Laboratory experiment
(In vitro model
system)
Pseudomonas
aeruginosa biofilms
M4 99.9% [137]
6K. SSEKATAWA ET AL.
Table 4. In vivo and ex vivo phage therapy experiments in animal models/tissues, fish, plants, poultry, piggery, and bees.
Field of application Phages Source of phages Level of application Target bacteria
Target bacteria strain/
pathotype
Level of
efficacy Ref.
Phage therapy in model organisms
Treatment of P. aeruginosa infection in
insect
PA5oct and KT28 Natural wastewater treatment plant In vivo-insect model P. aeruginosa PA PAO1 and 0038 93.60% [138]
Treatment of Burkholderia pseudomallei in
mice
Phage C34 Sea water In vivo-mouse model Burkholderia
pseudomallei
- 33.30% [139]
Treatment of S. aureus infection in BALB/C
mice
MR-10 - In vivo-mouse model S. aureus ATCC 43,300 (MRSA)
and ATCC 29,213
(MSSA)
100% [140]
Treatment of S. aureus osteomyelitis in
Rabbits
SA-BHU1, SA-BHU2, SA-BHU8, SA-BHU15
and SA-BHU21, SA-BHU37, SA-BHU47)
River, pond, and sewage In vivo-Rabbit model MRSA - 100% [58]
Treatment of GIT pathogenic E. coli
infection in white rats
EHEC-specific coliphage http://www.sumobrain.com/patents/wipo/
Methodsbacteriophage-design/WO201
0064044A1.pdf.
In vivo-mouse model E. coli. EHEC and non-EHEC
E. coli
99.9% [141]
Treatment of A. baumannii infection in
Mouse Model
BC62 of Myoviridae family Sewage water In vivo-mouse model Carbapenem resistant
A. baumannii
100% [34]
Treatment of A. baumannii pneumonia in
BALB/c mice
vB_AbaM-IME-AB2 (IME-AB2), Sewage In vivo-mouse model A. baumannii clinical
isolates (MDR and
sensitive)
- 100% [59]
Treatment of P. aeruginosa keratitis in
mice
фR18 and ФS12-1 Sewage In vivo-mouse model P. aeruginosa - 99.78% [142]
Prevention of V. cholerae infections in
mouse and rabbits
Vibrio phages ICP1, ICP2, and ICP3 Human feces In vivo-mouse and
rabbit models
V. cholerae AC 53, AC2846, and
AC4653
100% [143]
Treatment of S. Enteritidis infection in
Caenorhabditis elegans worms
ΦSP-1 and ΦSP-3 Chicken feces In vivo-worm model Salmonella enteritidis S49 94.8% [144]
Treatment of PDR A. baumannii infections
in Mice and human cells
Abp1 Sewage In vivo-mice model and
Ex vivo-human HeLa
cells
PDR A. baumanni - 100% [60]
Treatment of Pseudomonas aeruginosa
skin infections
Phage PA709 characterized Sewage water Ex vivo-human skin MDR P. aeruginosa MDR P. aeruginosa
709
99.99% [145]
Treatment of K. pneumoniae wound
infections in BALB/c mice
phage Kpn5 Sewage In vivo experiment Klebsiella pneumoniae B5055 100% [146]
Crop protection
Biocontrol of potato bacterial wilt P-PSG-3, P-PSG-4, P-PSG-1, P-PSG-8 to
P-PSG-12
water Field trial Ralstonia solanacearum PS-X4-1, PS-X10-2,
and PS-X13-1
80% in vivo
and 98%
in vitro
[147]
Biocontrol of alfalfa seeds spoilage
Salmonella enterica
Phages SSP5 and SSP6 sewage In vitro and in vivo
laboratory
experiments
Salmonella enterica S. oranienburg 0% [148]
Biocontrol of tomato bacterial wilt RsPod1EGY Soil In vitro and in vivo
laboratory
experiments
Ralstonia solanacearum K3, K9, K10,
K11, K12, K16, K17,
and K19
100% [149]
Phage biocontrol of antibiotics resistant
Dickeya dadantii which causes potato
tuber rot
Myoviridae family Caspian Sea water Laboratory experiment/
field Trial
Dickeya dadantii 100% in vitro
88.9% for
trial
[150]
Acquaculture
Aquaculture H20-Siphovirus and KVP40-Myovirus Sea water Laboratory experiment Vibrio anguillarum (BA35 and PF430-3) Reduced
biofilms
[151]
(Continued)
ALEXANDRIA JOURNAL OF MEDICINE 7
Table 4. (Continued).
Field of application Phages Source of phages Level of application Target bacteria
Target bacteria strain/
pathotype
Level of
efficacy Ref.
Phage therapy in model organisms
Aquaculture (treatment of ulcerative
lesions in catfish)
PA phages Waste water Field trial Pseudomonas
aeruginosa (MDR)
- 100% [152]
aquaculture VP-2 and VA-1 phage Sewage water Laboratory experiment
and trial
Vibrio. anguillarum 100% [13]
Aquaculture FpV-4, FpV-9, and FpV-21 pond water Laboratory Experiment Flavobacterium
psychrophilum
Reduced
bacterial
growth
[153]
Treatment of Vibrio parahaemolyticus
shrimp infections
V. parahaemolyticus phages (Myoviridae
family)
Shrimp pond water suspended sediment Laboratory experiment/
trial
Vibrio
parahaemolyticus
N1A and N7A 90% [154]
Phage therapy in poultry
In vivo phage therapy against
Campylobacter spp. infections in broiler
chicken
typeIII phages NCTC12672, 12,673, 12,674,
and 12,678 of the British phage typing
scheme
Lohmann Animal Health, GmbH. Field trial Campylobacter spp. 99% [155]
In vitro phage therapy against APEC Phage ØEC1 Chicken feces Laboratory experiment E. coli APEC O78:K80 99.99% [156]
Phage therapy in piggery
In vivo phage therapy of Staphylococcus
aureus nasal infection in pigs
phage K*710 and P68 Novolytics Ltd Trial using animal
model
Staphylococcus aureus
(MRSA)
ST398, spa type t011,
SCCmec type V
0% [71]
Phage therapy in apiculture
In vivo phage therapy of American
foulbrood caused by Paenibacillus
larvae
Siphoviridae (HB10c2) Glue-like liquid of a beehive Laboratory experiment
and field Trial
Paenibacillus larvae ERIC I DSM 7030 and
ERIC II DSM 25,430
2% [43]
8K. SSEKATAWA ET AL.
Table 5. Phage application in biosanitization.
Field of application Phage Phage source
Level of
application Target bacteria Bacteria strain/pathotype
Level of
efficacy Ref
Water and sewage treatment
Water purification vB_AspP-UFV1
(Podoviridae)
Sludge of wastewater Laboratory
experiment
A. soli, Pseudomonas sp., and Brevundimonas sp. AO1-02, AO2-07, AO1-30, and
AO1-33
Significant
biofilm
control
[157]
Coliform phage biocontrol in sewage Coliphage (Myovirus
and Podovirus)
River water Laboratory
experiment
E. coli E. coli SBSWF27 95.40% [158,159]
Biosanitization
Hospital anitizer Staphylococcal
phage and
Pyophage; GA,
Eliava Institute Laboratory
experiment
S. aureus, E. coli, and P. aeruginosa S. aureus (SA2-R73), E. coli (EC-
R60), and P. aeruginosa
(PAV6).
90% [86]
Hospital sanitizer AB1, AB2, AB6,
AB7 phages
Sewage or river water Laboratory
experiment
and trial
CR A. baumannii - 47.50% [87]
Hospital sanitizer Pyobacteriophag
polyvalent
Research and Production
Association “Microgen”
(Russia))
Trial staphylococci, streptococci, enterococci, proteus, klebsiella
(pneumoniae, and oxytoca), P. aeruginosa and E. coli
- 100% [88]
Phage cream and sanitizers Polyvalent Anti-
Staphylococcus
Phage K
ATCC 19,685-B1 Laboratory
experiment
and trial
MDR Staphylococcus aureus (MRSA and VRSA) - 100% [89]
Use of phages in semi solid creams for
control of Propionibacterium acnes
growth
PAC1 to PAC10 P. acnes strains isolated from
facial skin swabs
Laboratory
experiment
Propionibacterium acnes A1, A2, or E8 100% [90]
ALEXANDRIA JOURNAL OF MEDICINE 9
5. Data analysis
Data analysis was performed using Tukey’s multiple
comparisons test in STATA version 2018.1 to establish
whether; (a) the number of studies that reported
in vivo human phage therapy efficacy of 100% was
more pronounced than the number of studies that
recorded efficacy lower than 100%, (b) phages are
more efficient inhibitors of bacterial growth in ethanol
fermenters than phage endolysins, (c) there is
a considerable difference in in vitro phage therapy
outcomes against different species of clinical bacterial
isolates, (d) the outcomes of phage-mediated biocon-
trol in different fields are momentously dissimilar, (e)
MOIs used for ex vivo phage therapy/phage-mediated
biocontrol experiments, in vivo phage therapy and
in vitro phage therapy are soundly similar. A P value
of ≤ 0.05 indicated a significant statistical difference.
For comparison of phage therapy and phage-mediated
biocontrol efficacy across the different fields, only
fields that had three or more studies reporting phage
therapy efficacy in percentages were considered for
Tukey’s multiple comparisons test to prevent skewing
of data.
6. Results and discussion
6.1. Literature search
A total of 709 articles were generated through an
electronic database literature search conducted
between January and November 2018. The databases
were PubMed, Lib Hub, and Google Scholar, which
yielded 51, 416, and 242 articles, respectively.
Following the removal of duplications, 204 articles
were screened on the basis of their titles and abstracts.
Of the 204 articles; 90 did not meet the specified
inclusion criteria; and five full-text articles were not
accessible. Finally, 109 full-text articles were reviewed,
of which 95 full-text research articles fulfilled the
inclusion guidelines for this review, Figure 1. Studies
included in this review were grouped into in vivo
human phage therapy, in vivo phage therapy in
model organisms, phages as biocontrol agents in bio-
fuels fermentation, phages as biocontrol agents in
foods and beverages, in vitro phage therapy experi-
ments using clinical isolates, in vivo phage therapy in
crop protection, application of phages as biocontrol
agents in water purification, in vivo phage therapy in
aquaculture, in vivo phage therapy in apiculture,
in vivo phage therapy in a piggery in vivo and
in vitro phage therapy in poultry, application of phages
as bio-sanitizers, and in vitro use of phages as biocon-
trol agents in creams, Table 1–5, Figure 3.
Phage characterization; a prerequisite for phage-
mediated biocontrol of bacterial growth and in vivo
phage therapy
Phage-mediated biocontrol and phage therapy rely
on the ability of lytic phages to infect bacterial host
cells, hijacking the host metabolism and utilizing it to
produce their progeny. As a result, the lytic phages lyse
bacteria cells to release multiple phage virions which
spread to infect other host cells [10]. Contrary to that,
after infecting the bacterial host cells, lysogeny phages
incorporate their genetic material into the host gen-
ome resulting in their permanent existence as pro-
phages within host cells and all their offspring.
Figure 2. Comparison of mean MOIs between in vivo, in vitro, and ex vivo phage therapy. Tukey’s multiple-comparison test was
used to compute and compare MOIs P value of 0.0002 < 0.05 generated indicating significant variation between ex vivo/in vivo PT
and in vitro PT.
10 K. SSEKATAWA ET AL.
Phages neither replicate into virions nor lyse bacteria
throughout their lysogeny life time, hence called tem-
perate phages [10]. Furthermore, the integration of the
phage nucleic acids into its host bacterium protects the
temperate phage genome and has the ability to modify
the phenotype of the host bacterium cell [27].
Unfortunately, temperate phages might harbor toxin
encoding genes, virulent genes, and genetic determi-
nants of antibiotic resistance acquired from other bac-
terial hosts. Therefore, temperate phages may
transform the phenotype of the host bacteria and all
their progeny from avirulent/less virulent and antibio-
tic susceptible strains to highly virulent and antibiotic-
resistant strains [28,29]. Appropriately professionally
isolated and characterized phages must be used to
prevent horizontal gene transfer of undesirable genes
through phage-mediated biocontrol and phage ther-
apy [18,30,31]. Therefore, phages must be character-
ized morphologically by TEM and SDS PAGE protein
profiling to establish their families or if they are novel
phages followed by molecular characterization by
WGS to confirm their families and to detect any inte-
grase, toxin, and virulent genes in addition to antibio-
tic resistance genes by cross-referencing with known
phage genomes, virulent factors, toxin genes, and anti-
biotic-resistant genes libraries. A cheaper but less-
sensitive alternative to detect the presence of known
integrase gene, virulent factors (VF) and genetic deter-
minants of antibiotic resistance in phages is PCR
amplification using conventional integrase gene VF,
toxin genes, and antibiotic resistance genes primers.
However, PCR amplification has limitations as it will
not detect any possible novel VF and antibiotic resis-
tance genes harbored by phages hence making
molecular characterization of phages by WGS
a prerequisite prior to phage-mediated biocontrol of
bacterial growth and in vivo phage therapy [32,33].
However, only 12.6% (12) of the studies included in
this review conducted WGS. Bioinformatics analyses
and annotation demonstrated that myophages BɸC62
[34], DL52, DL60 and DL680 [35], DRA88 and phage
K [36], EcoInf [37], coliphagesɸAPCEc01, ɸAPCEc02
and ɸAPCEc03 [38], Phage P100 [39], leB, leE and leN
[40]:, podophages DL54, DL 62 and DL 64 [35]:, fHe-
Yen3-01 [41] and siphophages EcoSau [37], phSE-1,
phSE-2 and phSE-5 [42], fHe-Yen3-01, fHe-Yen9-01,
fHe-Yen9-02 and fHe-Yen9-03 [41] were safe to use
since they harbored no undesirable genes while
siphophage HB10c2 had a gene encoding a putative
beta-lactamase like protein [43]. Additionally, PCR
detected Stx I and II proteins encoding genes and
lysogeny module genetic determinants in phages
CB60P, MFA60N, CCO103, CBO103, and CCO113
[44], Table S1. If such phages are used in phage ther-
apy and phage-mediated biocontrol, they can facilitate
the horizontal flow of undesirable genes. This exorbi-
tantly underlines the importance of screening phages
using very sensitive tools like WGS. Nevertheless, only
36.8% (39) research articles included in this review
attempted to characterize phages; 3.2% (3) used PCR
to detect VFs and lysogeny modules while only 12.6%
(12) studies carried out WGS to fully illustrate the
phage genomes indicating that there is still a big gap
in ensuring phage therapy safety as per all the
reviewed articles that were in English, though all the
phages used for in vivo human phage therapy were
previously characterized by committed phage research
hubs. Furthermore, the morphology of phages was
Figure 3. Comparison of phage therapy (PT), phage-mediated biocontrol/diagnosis mean efficacy percentages. Tukey’s multiple-
comparison test was used to calculate and compare the mean percentage efficacies generating a P value of 0.148 > 0.05 after
exclusion of fields with less than three studies (water, piggery poultry, and apiculture).
ALEXANDRIA JOURNAL OF MEDICINE 11
determined by transmission electron microscopy
(TEM) in only 24.2% (23) studies. Basing on morphol-
ogy, the phages belonged to various families as follows:
Myoviridae; Siphoviridae; and Podoviridae in twenty,
nine and ten studies respectively. Whereas, one study
in each case reported phages as B1 morphology,
Phage-like particle, and Cytoviridae family, Table S1.
7. Phage stability
Establishing the abiotic conditions affecting phage activ-
ity and/or viability was done in 16.8% (16) studies. This is
an important criterion for selection since phage viability,
occurrence, and storage are affected by temperature, pH,
humidity, salinity, and other environmental conditions.
Deviation from the favorable physicochemical factors can
lead to the destruction of phages’ structural elements,
protein envelope, and loss of genetic material thereby
inactivating the phages [45,46]. These phages are isolated
from natural environments such as sewage, hospital, and
animal farm effluents, water bodies, foods, and beverages
and evaluated for in vitro, in vivo, and ex vivo phage
therapy and phage-mediated biocontrol where the pre-
vailing physicochemical factors are completely different,
Table 1–5. Hence, the need to establish the optimum
conditions for the highest phage efficacy. However, such
drawbacks can be mitigated by isolation of phages from
local geographical locations and similar hosts as for
in vivo phage therapy accompanied by assessing phage
stability via exposing them to different physicochemical
factors. Furthermore, during the preparation of commer-
cial phage-based remedy, physicochemical properties are
supposed to be investigated as they determine the shelf-
life of phages [47]. Despite that concern, only 9 (9.5%)
and 7 (7.4%) out of 95 research articles included in this
review evaluated the thermal and pH stability of phages,
respectively, Table S1. This partly explains why some
research articles reported very low or 0% phage efficacy
in in vivo studies.
8. Specicity of phages
Specificity restricts phage infections to only certain bac-
teria with corresponding receptors to which they can
bind; this determines the phage’s host range [48]. For
that reason, the application of phage therapy relies on an
accurate characterization of all the strains, pathotypes,
and serotypes of the target bacteria. Interestingly, if
phage therapy overcomes the current obstacles hindering
its approval universally, single phage and phage cocktail
formulations must be designed indicating the pharma-
ceutical dosage and the phage host range for a given
bacteria which calls for robust characterization of given
target host bacteria. Conversely, this review identified
gross deviation from the recommended procedure if
meaningful phage therapy outcomes are to be achieved
as only 55.8% (53) of studies reviewed attempted to use
identified bacterial host strains, serovars, and pathotypes,
Table 1–5. Worst still, no human in vivo phage therapy
trial reported characterization of the target bacteria to
their strains, pathotypes, and serotypes. Nevertheless, the
spectrum and efficacy of phages can be enhanced by the
use of phage cocktails. Phage cocktails also present
another advantage of preventing phage resistance [49,50].
9. Multiplicity of infection (MOI)
MOI is defined as PFU/CFU ratio [51]. MOI is an
imperative factor to be considered for prospective
phage therapy application. Increasing the PFU/CFU
ratio enhances the probability of phage particles
infecting their host bacteria. Therefore, in vivo and
ex vivo phage therapies require higher MOIs than
in vitro phage therapy as it is harder for phages to
locate and infect their hosts within living tissues, sur-
face of foods, and other materials being infected by
phages. Some studies recommend an MOI of over 100
for ex vivo and in vivo phage therapy and less than 10
for in vitro phage therapy [52]. This is in agreement
with the studies incorporated in this review that
reported MOI. The average MOI was highest in ex
vivo experiments (557,291.8), followed by in vivo
phage therapy (155,612.4) and in vitro phage biocon-
trol experiments had the lowest average MOI of 434.5
significantly different from ex vivo and in vivo MOIs,
Table S1 and Figure 2. Contrary to this, other studies
disregard the term MOI as it only describes the phage
quantities administered during dosing in relation to
the population of the target bacteria but does not put
into consideration the fact that; some phages fail to
penetrate tissues/materials and get inactivated before
adsorbing to the host cells, the host cell population is
liable to change before phage application, the bacterial
population may not easily be determined in case of
infections and physicochemical factors such as tem-
perature, pH, salinity, and humidity may inactivate
phages before adsorption. As a result, MOI input
may differ from the actual effective MOI [53].
Furthermore, to increase the prospect of phages
adhering and infecting their hosts; for experimental-
induced infections a very high MOI of >10
5
is recom-
mended [54] whereas in vivo phage therapy of natural
infection a very high titer value of > 1 × 10
8
PFU is
appropriate as bacterial host cells are lysed by simply
adsorption of phages before injection of their nucleic
acids into the host cells and replication [52,54].
However, phages are immunogenic when applied at
very high doses [55]; therefore, the host immune sys-
tem may identify and inactivate them. Additionally,
the MOI against biofilm infections should be higher as
indicated by the studies reviewed which compared
optimum MOI against bacterial suspension or free-
living bacteria to that against biofilms and/or immo-
bile bacteria, Table S1 and Figure 2. In in vitro
12 K. SSEKATAWA ET AL.
experiments, MOIs of 0.1, 1, and 10; and 100, 1,000,
and 10,000 [56]; 0.1 and 10 [36] were administered
against bacterial planktons and biofilms, respectively.
It is worth mentioning that in addition to high MOI,
the most suitable phages for phage-mediated manage-
ment of biofilm infections should encode polysacchar-
ide depolymerase which degrades the biofilm
polysaccharide matrix to ease phage interaction with
the host cells in the lower layers of the matrix [57].
10. Ecacy of phage therapy against drug
resistant and sensitive bacterial infections and
isolates
In vivo human phage therapy studies reported mixed
levels of efficacy ranging from 0% to 100%. The mode
and median efficacies were 100% while Tukey’s multiple
comparison test generated a P value of 0.009 < 0.05 indi-
cating that phage therapy efficacies of 100% were more
pronounced than efficacies lower than 100% in all the
in vivo human phage therapy. Interestingly, efficacies of
100% were scored when treating MRSA diabetic foot
ulcers, GIT MRSA infection, VRSA corneal abscess and
interstitial keratitis, and MDR Pseudomonas aeruginosa
UTI with phages. Furthermore, in vivo phage treatment
of MRSA osteomyelitis in Rabbits [58], carbepenem resis-
tant Acinetobacter baumanii infection in mice [34], MDR
Acinetobacter baumanii pneumonia in mice [59] and pan
drug resistant (PDR) Acinetobacter. baumannii infec-
tions in mice [60] provided 100% protection to model
animals against the super bugs while in vivo phage ther-
apy of MDR Pseudomonas aeruginosa ulcerative lesions
in catfish species achieved 100% success. It is also worth
noting that ex vivo phage therapy against MDR
Pseudomonas aeruginosa skin infections, MRSA biofilms
induced onto porcine skin burns, and PDR Acinetobacter
baumanni human HeLa cells infections recorded over-
whelming success. In vitro phage therapy against MRSA,
MDR Acinetobacter baumanni, MDR Pseudomonas aer-
uginosa scored an inhibitory efficacy ranging from 78% to
100% with an average of 95.4%. Data from around the
globe show an overall decline in the total reserves of
antibiotics efficacy: resistance to all first-line and last-
resort antibiotics is increasing [3]. For instance, in sub-
Saharan Africa, India, Latin America, and Australia,
MRSA incidence is still intensifying [3,6162], and esti-
mated at 47% in India in 2014, and 90% in Latin
American hospitals in 2013 [61][]. MRSA causes
35–46% of wound complication in Mulago referral hos-
pital [63,64]. The increased prevalence of community
acquired E. coli isolates coding for extended-spectrum
beta lactamases competent of hydrolyzing approximately
all beta lactams antibiotic except carbapenems has been
reported globally [65]. In more than a decade, carbape-
nem resistance in Enterobacteriaceae bacteria has been
observed yet Carbapenems such as imipenem, ertape-
nem, meropenem, and doripenem are the newest
synthesized molecules with the broadest spectrum of
activity and consequently considered the first-line ther-
apy antibiotics in the treatment of multi-resistant gram-
negative bacterial infections [66,67]. The magnitude of
MDR Pseudomonas aeruginosa and Acinetobacter bau-
mannii is a great threat to the health sector worldwide
[68]. The promising outcomes of in vivo, ex vivo, and
in vitro phage therapy of MDR bacterial infections and
isolates exhibit that phage therapy if employed appropri-
ately is more effective than antibiotics and therefore can
replace or supplement antibiotics as a routine in the
management of both resistant and sensitive bacterial
infections. However, limited success was attained when
treating S. aureus and P. aeruginosa wound infection in
humans, acute human E. coli infections, MRSA nasal
infections in pigs and American foulbrood caused by
Paenibacillus larvae [43,69–71]. This is in contrary to
the in vitro experiments carried out in two of the studies
where total eradication of the bacteria was achieved
[43,71]. This can be attributed to the change in physiolo-
gical conditions: loss of phage viability due to deviation
from their optimum temperature and pH in unnatural
environments [46].
11. Endolysins versus phage particles
Phages code tail spike proteins for identification and
adhesion to receptors on the host cell surface. The tail
spikes proteins are often incorporated with peptidoglycan
hydrolases that locally hydrolyze the bacterial cell wall
peptidoglycan, thus creating an opening for injection of
phage nucleic acids which marks the initiation of the
infection process [72]. An additional type of phage-
derived enzymes; the peptidoglycan hydrolases called
endolysins degrade the peptidoglycan liberating the pro-
geny virions from the host cell at the end of the lytic phage
cycle [73]. Gram-positive bacteria do not possess
a shielding outer layer thereby making exogenous appli-
cation of endolysins achieve speedy and effective lysis.
This property makes endolysins promising possible alter-
native antimicrobial agents [23]. Several studies have
reported endolysins as potential therapeutic agents with
high efficacy and safety [74]. In addition, endolysins
possess an added advantage over conventional antibiotics
as; they exhibit great specificity exerting selective pressure
on target pathogenic bacteria populations [75,76], emer-
gence of resistance against endolysins is implausible given
that phage (endolysins) coevolve with their host bacteria,
the host receptor site where endolysins bind are highly
conserved thereby making their alteration highly detri-
mental to the host bacterium [76,77]. Furthermore, endo-
lysins degrade the cell wall externally without the burden
of entering the bacterial cell hence evading the common
antibiotic resistance mechanisms such as the active efflux
pump and decreased membrane permeability [78]. A lot
of ethical and safety concerns have been vehemently
expressed about the use of live viruses as therapeutics in
ALEXANDRIA JOURNAL OF MEDICINE 13
the treatment of bacterial infections; currently, the
immediate hope lies in the use of phage endolysins in
the near future to combat the increasing antibiotic resis-
tance. Fortunately, to meet the high demand, endolysins
can be produced using recombinant DNA technology
[79–81]. This review compared the use of phages and
endolysins to suppress bacterial growth during ethanol
fermentation. Phages demonstrated superior efficacy
than recombinant phage endolysins with mean efficacy
of 99.5% for phages and 83.6% for phage endolysins but
not significantly divergent as revealed by one-way
ANOVA (P value of 0.13 > 0.05). This clearly supports
the use of phages and endolysins hand in hand as ther-
apeutic agents.
12. Application of phages in Biosanitization
and Biopreservation
Infectious food and water-associated diseases are the
major causes of mortality and morbidity worldwide
[6,7]. Irrational use of antibiotics in livestock has resulted
in antibiotic resistance which spillover to humans
through contaminated food, water, and environment [-
3–5,67]. Fortunately, in 2006 the US Food and Drug
Administration (FDA) approved the utilization of 6 inde-
pendently purified LMP-102 phages as biopreservative
antimicrobial agents in RTE meat and poultry products
against Listeria monocytogenes [82]. In this review, the
literature search yielded 21 (22.1%) research articles
reporting foods and beverages phage-mediated bio-
preservation with average, mode, median efficacy of
96.5%, 100%, and 100%, respectively. In a water deconta-
mination study, phages eradicated 95.4% of the coliform.
This is a clear indicator of the potency of phages as bio-
preservative and bio-decontamination agents and conse-
quently their approval to preserve food and decontami-
nate water following robust characterization should be
considered to prevent transmission of antibiotic-resistant
and susceptible food and water-associated infection.
Furthermore, the hospital environment polluted by
infected patients with antibiotic-resistant bacteria is
incriminated as the main route of transmission
[83,84]. This has been a result of the emergence of
bacterial resistance to the conventional disinfectants
[83]. The possibility of a horizontal flow of mobile
genetic elements encoding antibiotic resistance from
clinical to environmental bacteria within the hospital
is high hence advancing the evolution of new antibiotic-
resistant bacterial strains [85]. On a good note, bio-
disinfection using phages as demonstrated by this
review is promising: for instance, phage-mediated bio-
sanitization eradicated 90% of Staphylococcus aureus,
Escherichia coli, and Pseudomonas aeruginosa tainted
on plastic, glass, and ceramic materials mimicking hos-
pital surfaces [86] while phage-mediated sterilization
trial of the ICU reduced the prevalence of carbapenem-
resistant Acinetobacter baumanii by 47.5% [87]. In
another phage sanitization trial, phages completely
eliminated staphylococci, streptococci, enterococci, pro-
teus, Klebsiella pneumoniae, Klebsiella oxytoca,
Pseudomonas aeruginosa, and Escherichia coli from the
hospital environment [88] while phage-based sanitiza-
tion cream completely inhibited MRSA and
Propionibacterium acnes growth [89,90]. With those
laudable bio-sanitization results, the use of phages to
complement conventional disinfection strategies could
exhibit valuable outcomes.
13. Phages and endolysin as alternative
antibacterial decontamination agents
Lactic acid bacteria (LAB) are by far the commonest
bacterial contaminants of biofuel production facilities
and are believed to hamper the ethanol fermentation
process hence limiting ethanol production. Ethanol fer-
mentation presents an environment of high ethanol con-
centration, low pH, and low oxygen concentration
thereby favoring the growth of Lactobacillus sp which
are well adapted to survive under such conditions.
Currently, there is no appropriate strategy to combat
ethanol loss due to LAB contamination as all possible
measures have limitations [91]. Contrary to that, the four
experimental studies which employed phages and endo-
lysins to control LAB growth during ethanol fermenta-
tion analyzed in this review demonstrated eye-catching
bacterial growth suppression outcomes with mean effi-
cacy of 91.6%. Most importantly, phage and endolysins
mediated ethanol fermentation facility decontamination
restored normal ethanol yield without losing their viabi-
lity [37,92]. Because of the promising results, to eliminate
the use of antibiotics for decontamination in the ethanol
fermentation business, phages and endolysins should be
considered as alternatives.
14. Limitations
Hypothetically, all bacteria can be lysed by at least one
type of bacteriophage. In the light of this, phages are
considerably more efficacious than antibiotics.
However, phage antibacterial applications have limita-
tions. Most phages have demonstrated a broad spectrum
hence can lyse both the target pathogenic strains and
potentially beneficial bacterial strains. Additionally, it is
difficult to isolate phages without any undesirable genes
such as antibiotic-resistant genes, bacterial virulent genes,
and integrase genes. Phages with such genes may con-
tribute to the development of highly pathogenic antimi-
crobial-resistant bacteria. Furthermore, phage-based
therapeutic formulation and stabilization is still
a challenge as previous studies reported that the stability
of phage formulations for clinical use is stringently influ-
enced by the phage type. Thus, each phage type requires
its unique stabilization strategy and this is extremely
complicated for phage cocktail formulations. The
14 K. SSEKATAWA ET AL.
evolution of bacterial resistance against phages mainly
mediated by loss or alteration of the bacterial phage
receptors and bacterial secretions that prevent phage
adsorption has been implicated as another limitation
affecting phage therapy. Inactivation of phages by the
immune system has also been reported as a drawback of
phage therapy.
15. Conclusion
The high prevalence of MDR infections has resulted in
familiar bacterial diseases becoming difficult to treat.
Moreover, hospital-associated infections (both sensitive
and MDR) are mainly acquired through contaminated
surfaces and medical equipment. However, phage-
mediated bio-sanitization, in vivo, ex vivo, and in vitro
phage therapy experiments and trials analyzed by this
review showed that phages can mitigate the burden
caused by MDR infections and contamination of hospital
surfaces as well as medical devices. Furthermore, water
and food-borne bacterial infections have been implicated
as the major cause of mortality and morbidity globally
and LAB as the main cause of yield loss in the biofuels
fermentation industry. Analysis of phage/endolysin
mediated bio-preservation and bio-decontamination stu-
dies by this review showed that phages and endolysins
were highly effective. Thus, phage technology presents an
opportunity for developing alternative therapeutic, bio-
preservative, bio-decontamination, and bio-sanitization
approaches. Despite the undisputable efficacy of phage
therapy and phage-mediated biocontrol, rigorous inves-
tigations using highly sensitive techniques should be car-
ried out to ensure that only appropriate professionally
lytic and safe phages are used. Thus, for low- and middle-
income countries, there is a need to develop affordable
and appropriate methods for screening of phages for
undesirable genes. Moreover, the challenge of immuno-
genicity that may be associated with in vivo application of
phages needs to be explored further.
Acknowledgements
We are thankful to MAPRONANO ACE for funding this work.
Disclosure statement
The authors declare that they have no competing interests.
Funding
The authors declare that this review article was funded by
Africa Centre of Excellence in Materials, Product
Development & Nanotechnology; MAPRONANO ACE
Makerere University;African Center of Excellence in
Materials, Product Development and Nanotechnology,
College of Engineering Design, Art and Technology
Makerere University [P151847IDA credit 5797-UG].
Notes on contributors
Kenneth Ssekatawa is a PhD candidate at Makerere
University and Lecturer Department of Biochemistry,
Kampala International University. Kenneth specializes in
molecular microbiology, antimicrobial resistance and
nanobiotechnology.
Prof. Denis K. Byarugaba He is a Professor of Microbiology,
College of Veterianary Medicine Animal Resources and
Biosecurity, Makerere University. He is currently involved
in research on influenza viruses and highly pathogenic
pathogens with potential for causing pandemic threats and
antimicrobial resistance.
Dr. Charles D. Kato is a lecturer at college of Veterinary
Medicine Animal Resources and Biosecurity. Charles'
research interests are in biomark research regarding disease
co-infection and host-parasite interactions in infectious and
zoonotic diseases. Charles is conducting research in phage
mediated therapy.
Dr. Eddie M. Wampande currently works as a senior lec-
turer College Veterinary Animal Resources and Biosecurity,
Makerere University. He specializes in Microbiology and
Parasitology.
Prof. Francis Ejobi is a Professor of Microbiology, Makerere
University specializing in infectious disease, epidemiology
and antimicrobial resistance
Prof. Robert Tweyongyere Robert is a research fellow at the
Uganda Virus Research Institute, Entebbe and a Senior
Lecturer at College of Veterinary Medicine Animal
Resources and Biosecurity Makerere University. His
research interests are in infectious diseases.
Dr. Jesca L. Nakavuma Jesca works as a senior lecturer and
microbiologist, Department of Biomolecular and
Biolaboratory Sciences, College of Veterinary Medicine
Animal Resources and Biosecurity, Makerere University.
She is the Founder Chair Phage Team Uganda and currently
working on a project aimed at using phage therapy to con-
trol and treat bacterial infections in fish.
ORCID
Kenneth Ssekatawa http://orcid.org/0000-0003-4061-
9345
Ethics and consent to participate:
Not applicable
Consent for publication:
Not applicable
Availability of Data and Materials:
Supplementary data summarized in tabular form have been
submitted with the manuscript.
Authors’ contributions
This work was carried out in collaboration between all
authors. Jesca L. Nakavuma (JLN), Dennis K Byarugaba
ALEXANDRIA JOURNAL OF MEDICINE 15
(DKB), Robert Tweyongyere (RB), and Francis Ejobi (FB)
conceptualized this project and designed the format for this
review. Kenneth Ssekatawa (KS), Edward Wampande (EW),
Charles Kato Drago (CKD) & JLN performed the literature
search and data analysis. All authors drafted the section of
the literature review. KS, JLN, and CKD wrote the first draft
of the manuscript and managed manuscript revisions. All
authors read and approved the final manuscript.
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