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Citation: Huang, Y.; Wang, W.;
Zhang, Z.; Gu, Y.; Huang, A.; Wang,
J.; Hao, H. Phage Products for
Fighting Antimicrobial Resistance.
Microorganisms 2022,10, 1324.
https://doi.org/10.3390/
microorganisms10071324
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Received: 27 May 2022
Accepted: 27 June 2022
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microorganisms
Review
Phage Products for Fighting Antimicrobial Resistance
Yuanling Huang 1,2, Wenhui Wang 1,2, Zhihao Zhang 1,2, Yufeng Gu 1,2, Anxiong Huang 1,2 , Junhao Wang 1,2
and Haihong Hao 1,2,3,4,*
1National Reference Laboratory of Veterinary Drug Residues, Huazhong Agricultural University,
Wuhan 430070, China; huangyuanling0226@163.com (Y.H.); wwh6572@163.com (W.W.);
zhangzhihao@webmail.hzau.edu.cn (Z.Z.); guyufeng@webmail.hzau.edu.cn (Y.G.);
anxionghuang@webmail.hzau.edu.cn (A.H.); wangjunhao@webmail.hzau.edu.cn (J.W.)
2MOA Laboratory for Risk Assessment of Quality and Safety of Livestock and Poultry Products,
Huazhong Agricultural University, Wuhan 430070, China
3Shenzhen Institute of Nutrition and Health, Huazhong Agricultural University, Shenzhen 518000, China
4
Shenzhen Branch, Guangdong Laboratory for Lingnan Modern Agriculture, Genome Analysis Laboratory of
the Ministry of Agriculture, Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural
Sciences, Shenzhen 518000, China
*Correspondence: haohaihong@mail.hzau.edu.cn
Abstract:
Antimicrobial resistance (AMR) has become a global public health issue and antibiotic
agents have lagged behind the rise in bacterial resistance. We are searching for a new method
to combat AMR and phages are viruses that can effectively fight bacterial infections, which have
renewed interest as antibiotic alternatives with their specificity. Large phage products have been
produced in recent years to fight AMR. Using the “one health” approach, this review summarizes
the phage products used in plant, food, animal, and human health. In addition, the advantages
and disadvantages and future perspectives for the development of phage therapy as an antibiotic
alternative to combat AMR are also discussed in this review.
Keywords:
antimicrobial resistance; phage products; advantages and disadvantages of phage therapy;
development prospects
1. Introduction
Antimicrobial resistance is a naturally evolving phenomenon that emerged soon after
the discovery of penicillin in 1940 [
1
]. Antibiotics are highly efficient against bacterial
infections, saving millions of lives and drastically reducing mortality rates. However,
multidrug-resistant bacteria (MDR), extensively drug-resistant bacteria (XDR), and even
pan-resistant bacteria (PDR) have evolved as a result of antibiotic overuse, abuse, and
misuse. In particular, ESKAPE bacteria seriously threaten human health worldwide. Ac-
cording to the latest estimates, approximately 700,000 people worldwide die directly from
AMR bacteria each year, with that number possibly rising to 1 billion by 2050 [
2
]. AMR
is one of the top ten global public health threats facing humans, according to the WHO.
As a result, FAO, WOAH, and WHO attach great importance to this and jointly launched
the “one health” approach to combat AMR [
3
]. The interdependent relationship between
the food chain and the environment makes resistant bacteria widespread in plants, ani-
mals, food, and humans, and the “one health” approach trinity model is ideally suited to
address AMR [
4
,
5
]. Phages are currently one of the antibiotic alternatives with the most
potential because of their ability to effectively combat bacterial infections. Phages are a new
alternative therapy under the “one health” approach that can be used to control bacteria in
plants, animals, food, and humans [
6
]. Currently, phage therapy is emerging globally, and
in this review, we summarize the application of phage products for plants, animals, food,
and human health from the perspective of “one health” from the two databases of phage
companies and bacteriophage news [
7
,
8
]. Furthermore, the advantages and disadvantages
Microorganisms 2022,10, 1324. https://doi.org/10.3390/microorganisms10071324 https://www.mdpi.com/journal/microorganisms
Microorganisms 2022,10, 1324 2 of 22
of phages as antibiotic alternatives to combat AMR and their future development prospects
are also discussed in detail.
2. Phage Biology
As early as 1896, Ernest Hankin discovered antibacterial substances against
Vibrio cholerae
from water extracted from the Ganges and Jumna rivers in India, laying the foundation
for the subsequent discovery of phages [
9
,
10
]. The term “phage” was introduced by
Félix d’Hérelle after he discovered the “anti-microbe” Shigella in 1917 [
11
]. Phages are
abundant entities on the planet, with a population of 10
31
, which is 10–100 times that
of their obligatory parasitic host bacterium [
12
]. The genome of phages is composed of
single-stranded (ss) or double-stranded (ds) DNA or RNA, which is encapsulated by a wide
variety of protein capsids. The universal viral taxonomy established by the International
Committee on Taxonomy of Viruses (ICTV) divides phages into polyhedral, filamentous,
pleomorphic, and tailed according to capsid morphology [
13
]. Phages can be classified into
temperate and virulent based on their life cycle and reproductive characteristics. However,
the process of bacterial infection is different between temperate and virulent phages.
Virulent phages enter the lytic cycle, which usually consists of five stages. The tail
filament first adsorbs to a specific receptor on the surface of the host bacteria. These
receptors can be located on cell walls, capsular polysaccharides, outer membrane proteins,
efflux pumps, or appendages, such as pili and flagella [
11
] (Figure 1A). Second, the phage-
derived enzymes (such as endolysins) lyse the peptidoglycan of the cell wall and the tail
pipe penetrates through the cell membrane to inject its DNA into the host bacteria [
14
].
Third, phages perform biosynthesis, such as nucleic acid replication, RNA transcription,
and protein translation in dormitory cells. Fourth, phages assemble into progeny phages.
Finally, when the number of progeny phages reaches a certain threshold, bacteria lyse and
release progeny phages [
15
] (Figure 1B). For Gram-negative bacteria, lysis is achieved by
three different functional proteins, holins, endolysins, and spanins, which act on the inner
membrane, peptidoglycan, and outer membranes of the cell envelope, respectively [
16
].
Temperate phages differ from virulent phages in a number of important ways. For example,
temperate phages integrate their genomes into the chromosomes of host bacteria, which do
not lyse but enter the lysogenic cycle. Phages grow and multiply with host bacteria [
17
].
Under certain conditions, temperate phages can also enter the lytic cycle, depending mainly
on phage-encoded repressors and regulators, as well as the control of phage enzymes [
18
].
For example, under stress responses and light, temperate phages can initiate the expression
of lytic genes. Temperate phages can regulate the gene expression and behavior of bacteria
through different mechanisms and enhance phage-host fitness [
19
]. In addition, both
virulent and temperate phages have a pseudo-lysogenic nature, which means that viral
DNA is present in the host bacteria in a form similar to a plasmid and the host at this
moment is only the vector of the phage [14,18].
The key to killing bacteria by phages depends on lysing the bacterial cell wall and
virulent phages are generally selected for treatment. It has commonly been assumed that
icosahedral DS DNA phages containing tails can effectively treat human and animal infec-
tions [
20
]. Bacteria may acquire resistance genes or genes with pathogenic potential after
the lysogenic transformation of temperate phages and they are generally not recommended
for therapeutic purposes [
11
]. However, with advances in synthetic biology, temperate
phages can be designed to interfere with bacterial intracellular processes and cause bacte-
rial cell death. Alternatively, genomes of temperate phages were engineered to eliminate
known virulence genes involved in the lysogenic cycle [
21
]. The current crisis of AMR
makes phage therapy re-emerge globally and the cases of phage therapy in preclinical
research are also gradually increasing [22–24].
Microorganisms 2022,10, 1324 3 of 22
Microorganisms2022,10,xFORPEERREVIEW3of20
Figure1.Mechanismofphageinfestationofbacteria.(A)Receptorsforphageadsorptiononbacte‐
ria(purplerepresentsporins,bluerepresentseffluxpumps,yellowrepresentsflagella,redrepre‐
sentspili,andgrayrepresentscapsularpolysaccharide)(B)Describedfivephasesofaphagelytic
cycleandalysogeniccycle.
3.PhagesProductsinPlantHealth
Morethan200plantbacteriahavebeenreportedtocausesignificantcroplosses
duringpreharvest,storage,andtransport[25].Antibioticshavealsobeenusedagainst
plantpathogenssinceWorldWarII,andAMRhasbeenwidespreadinsomeplantsand
cropsduetothedisseminationofresistancegenesintheenvironment.Forexample,an‐
tibioticresistancegenes(strAB)haveemergedinPseudomonassyringae,Xanthomonas
campestris,andErwiniaamylovora,triggeringresistancetostreptomycin[26].Thefirstex‐
perimentalevidencethatphagesmaybeassociatedwithplantpathogenicbacteriawas
thediscoverythatfiltratesobtainedfromcabbagewereabletoinhibitcabbagedecay
causedbyXanthomonascampestrispv.[27].Subsequently,in1925KotilaandCoonsused
phagestopreventsoftrotcausedbyPectobacteriumatrosepticumandPectobacteriumca‐
rotovorumsubsponpotatotuberandcarrotslices,respectively.In1935,Thomasused
phageagainstthephytopathogenPantoeastewartiitosignificantlyreducetheincidence
[28].Modernstudieshaveshowntheeffectivenessofphagesforplanthealthandcan
targetdrug‐resistantplant‐bacteriawithextremelyhighefficiency.Forexample,theiso‐
lationofanovelphageXoo‐sp2infectedwithXanthomonasoryzaefromsoilcaneffective‐
lycontrolbacterialblightinrice[29].TheengineeredphageY2caneffectivelycontrol
andrapidlydetectErwiniaamylovora,afireblightpathogen[30].Recently,threephage
cocktails(φEa2345‐6,φEa1337‐26,andEh21‐5)areeffectiveagainstfireblightinapples
andpears.Fourphagecocktails(Eram2,Eram26,Eram24,andEram45)areeffective
againstfireinblightedpears[31].Xylellafastidiosa(Xf)isanovelplantpathogenwitha
widerangeofplanthostsandaspectrumofinsectspeciesthatarenowcausingsignifi‐
cantdamagetoworldagriculture.Phageisanoveltherapytocontroldiseasescausedby
Xf[32,33].GiventhecurrentinvasionofXfintoagroecosystems,thesephagescanbe
implementedasbiologicalagentsandareexcellentcandidatesfordevelopmentinto
phagecocktails.
Asphagestudiesadvance,thenumberofphageproductstargetingplantpathogens
inthemarketisalsoincreasing.Atpresent,theUnitedStatesEnvironmentalProtection
Agency(USEPA)hasapprovedseveralphageproductstofightplantpathogens,and
Figure 1.
Mechanism of phage infestation of bacteria. (
A
) Receptors for phage adsorption on bacteria
(purple represents porins, blue represents efflux pumps, yellow represents flagella, red represents
pili, and gray represents capsular polysaccharide) (
B
) Described five phases of a phage lytic cycle
and a lysogenic cycle.
3. Phages Products in Plant Health
More than 200 plant bacteria have been reported to cause significant crop losses dur-
ing preharvest, storage, and transport [
25
]. Antibiotics have also been used against plant
pathogens since World War II, and AMR has been widespread in some plants and crops
due to the dissemination of resistance genes in the environment. For example, antibiotic
resistance genes (strAB) have emerged in Pseudomonas syringae,
Xanthomonas campestris,
and Erwinia amylovora, triggering resistance to streptomycin [
26
]. The first experimental
evidence that phages may be associated with plant pathogenic bacteria was the discov-
ery that filtrates obtained from cabbage were able to inhibit cabbage decay caused by
Xanthomonas campestris pv. [27].
Subsequently, in 1925 Kotila and Coons used phages to
prevent soft rot caused by Pectobacterium atrosepticum and Pectobacterium carotovorum subsp
on potato tuber and carrot slices, respectively. In 1935, Thomas used phage against the
phytopathogen Pantoea stewartii to significantly reduce the incidence [
28
]. Modern studies
have shown the effectiveness of phages for plant health and can target drug-resistant plant-
bacteria with extremely high efficiency. For example, the isolation of a novel phage Xoo-sp2
infected with Xanthomonas oryzae from soil can effectively control bacterial blight in rice [
29
].
The engineered phage Y2 can effectively control and rapidly detect Erwinia amylovora, a fire
blight pathogen [
30
]. Recently, three phage cocktails (
ϕ
Ea2345-6,
ϕ
Ea1337-26, and Eh21-5)
are effective against fire blight in apples and pears. Four phage cocktails (Eram2, Eram26,
Eram24, and Eram45) are effective against fire in blighted pears [
31
]. Xylella fastidiosa (Xf) is
a novel plant pathogen with a wide range of plant hosts and a spectrum of insect species
that are now causing significant damage to world agriculture. Phage is a novel therapy to
control diseases caused by Xf [
32
,
33
]. Given the current invasion of Xf into agroecosystems,
these phages can be implemented as biological agents and are excellent candidates for
development into phage cocktails.
As phage studies advance, the number of phage products targeting plant pathogens
in the market is also increasing. At present, the United States Environmental Protection
Agency (USEPA) has approved several phage products to fight plant pathogens, and com-
mercial phage products are summarized in Table 1. Only six phage products for plant health
have been commercialized, mostly for soft rot Enterobacteriacea,
Clavibacter michiganensis,
Microorganisms 2022,10, 1324 4 of 22
Xanthomonas citri, and Erwinia amylovora, which are all prevalent plant pathogens. Om-
niLytics (Kuala Lumpur, MY, USA) was the earliest registered phage-based biopesticide
product and its AgriPhage
™
product line has been approved by the USEPA for the control
of bacterial diseases in citrus, tomato, apple, and pear. In addition, the product also obtains
an Organic Materials Review Institute (OMRI) listing for commercial organic growers.
Enviroinvest Erwiphage PLUS (Hungary) is the second company to obtain a biopesticide
registration and its product Erwiphage can fight fire blight caused by Rosaceae plants.
The Biolyse
®
BP product, developed by APS Biocontrol (Dundee, UK), is a phage-based
potato tuber wash for the prevention of soft rot during storage. Fixed-Phage (Glasgow,
UK) also has a product for a variety of bacteria (agriPHIX
™
) that plays a significant role in
improving the storage of a range of crops. In conclusion, there are a few phage products
for plants that need to be further developed.
Table 1. Commercial phage products for plant health.
Target Bacteria Company Products Regulatory
Approval Certifications Application Ref.
Soft rot
Enterobacteriacea
APS Biocontrol
Ltd. (SCO) Biolyse®BP Approved UK, European
Food processing assistants
in the potato packaging
industry
[34]
Clavibacter
michiganensis
OmniLytics Inc.
(USA)
AgriPhage
CMM™
EPA approved USA, Canada
Tomato bacterial canker [35,36]
Xanthomonas citri AgriPhage™
Citrus Canker Citrus canker
[27]
Erwinia amylovora
AgriPhage™
Fire Blight
Fire blight for apples
and pears
Erwiphage PLUS
(HU) Erwiphage
Undefined
Hungary
Fire blight caused by plants
in the rose family [37]
Variety of bacteria
Fixed-Phage (UK)
agriPHIX™ UK Effective improvement of
storage for a range of crops
[38]
4. Phage Products in Animal Health
The indiscriminate and extensive use of antibiotics in animals has been one of the prin-
cipal reasons for the rapid spread of AMR. The new EU law prohibiting the prophylactic
use of antibiotics in farmed animals was implemented in 2022 and the use of antibiotics is
also strictly regulated in the United States and Canada [
39
]. The recent resurgence of phage
therapy has also prompted the extensive application of phages in veterinary medicine. The
first known therapeutic use of phages in veterinary medicine was associated with Felix
d’Herelle, who used phages in 1919 to prevent and treat Salmonella infections in chickens
and effectively reduce mortality in chickens [
40
]. However, when Pyle used phage therapy
in 1926 to treat Salmonella Enteritidis infection in chickens, the results were less than encour-
aging [
41
]. Until the 1980s, William Smith reconsidered the use of phage therapy in animals
and experimented with chickens, cattle, and pigs [
42
]. The early British team conducted a
small clinical trial of a phage cocktail for canine otitis media caused by P. aeruginosa in 2010
and the results were greatly encouraging [
43
]. Subsequently, the application of phages in
animals has been increasing, mainly for treating E. coli and Salmonella infections in poultry
and pigs, as well as mastitis in cattle caused by S. aureus [
44
]. In aquaculture, phages are
also effective against Vibrio,Pseudomonas, and Aeromonas, reducing fish mortality [45].
The preharvest application of phages can effectively reduce the infection and coloniza-
tion of live poultry and minimize the risk of pathogens entering the food chain, thereby
reducing the infection of zoonotic bacteria [
46
]. At present, phage products in animals
mainly focus on the preharvest application of livestock, poultry, and the clinical appli-
cation of pets. The detailed commercial phage products used in animals are shown in
Table 2. A total of 9 of the 38 veterinary phage products counted have been approved by
the FDA and 3 by the European EFSA. The products developed by Intralytix (Baltimore,
Microorganisms 2022,10, 1324 5 of 22
AR, USA) mainly focus on pet food safety and preharvest intervention. The product line of
PhagePharm (Qingdao, China) focuses on common pathogens in the environment and is
used as an environmental improver, and the product line of Fixed-Phage (UK) is mostly
phage cocktails. Figure 2A reveals that 20 mono-component phage products in the market
almost target E. coli, Salmonella, and C. perfringens in poultry. A variety of phage cocktail
products also target the infection of these bacteria. Most veterinary phage products are
mainly in the form of food additives in animal feed or drinking water to prevent and
control bacterial diseases. Only a few phage products are made into gel formulations
for topical epidermic medication, such as Staphage Lysate (SPL)
®
, a phage product from
Delmont Laboratories (Swarthmore, PA, USA), which is also the only staphylococcal product
approved for use in Staphylococcus canis skin infections [
47
]. In contrast, phage products
targeting companion animals remain to be further investigated, especially the study of
bacterial dermatology, which may have significance in the future.
Microorganisms2022,10,xFORPEERREVIEW5of20
bytheFDAand3bytheEuropeanEFSA.TheproductsdevelopedbyIntralytix(Balti‐
more,AR,USA)mainlyfocusonpetfoodsafetyandpreharvestintervention.Theprod‐
uctlineofPhagePharm(Qingdao,China)focusesoncommonpathogensintheenvi‐
ronmentandisusedasanenvironmentalimprover,andtheproductlineofFixed‐Phage
(UK)ismostlyphagecocktails.Figure2Arevealsthat20mono‐componentphageprod‐
uctsinthemarketalmosttargetE.coli,Salmonella,andC.perfringensinpoultry.Avariety
ofphagecocktailproductsalsotargettheinfectionofthesebacteria.Mostveterinary
phageproductsaremainlyintheformoffoodadditivesinanimalfeedordrinkingwa‐
tertopreventandcontrolbacterialdiseases.Onlyafewphageproductsaremadeinto
gelformulationsfortopicalepidermicmedication,suchasStaphageLysate(SPL)®,a
phageproductfromDelmontLaboratories(Swarthmore,PA,USA),whichisalsotheon‐
lystaphylococcalproductapprovedforuseinStaphylococcuscanisskininfections[47].In
contrast,phageproductstargetingcompanionanimalsremaintobefurtherinvestigated,
especiallythestudyofbacterialdermatology,whichmayhavesignificanceinthefuture.
Figure 2.
Analysis of phage products from the perspective of the “one health” approach. (
A
) Targeted
bacteria for mono-component phage products in animals. (
B
) Targeted bacteria for phage products in
food processing. (
C
) Targeted bacteria for mono-component phage products in humans. (
D
) Main
dosage forms of phage products in human therapy. (
E
) Main routes of administration for human
phage products.
Microorganisms 2022,10, 1324 6 of 22
Table 2. Commercial phage products for animal health.
Target Bacteria Company Products Regulatory
Approval Certifications Application Ref.
E. coli
Intralytix (USA)
Ecolicide®
FDA USA
For E. coli O157:H7
contamination in Pet Food [48]
Ecolicide
PX™
For E. coli O157:H7
contamination on animal fur
Arm and
Hammer Animal
& Food
Production (USA)
Finalyse®USDA, FSIS USA
A preharvest antimicrobial
hide wash used to reduce E.
coli O157:H7
[34]
Proteon
Pharmaceuticals
(POL)
BAFACOL™ EFSA Poland Feed additive to prevent
pathogenic E. coli in poultry. [49]
Phagelab (CHI) Swine
product Undefined Chile Liquid Food Additive
Eliminates E. coli in Swine [50]
Salmonella
Intralytix (USA)
SalmoLyse®
FDA USA
Salmonella Contamination in
Pet Food [51,52]
PLSV-1™ Salmonella Contamination
in Poultry [44,53]
Proteon
Pharmaceuticals
(POL)
BAFASAL+G
®EFSA Poland Feed additive to treat the
digestive tract of poultry [51,54]
UniFAHS SalmoGuard FDA
Southeast
Asian
countries
Poultry feed additives [52,55]
OmniLytics Inc.
(USA) BacWash™ USDA USA For Hides of livestock
surface disinfection [56]
SciPhage (CO) SalmoFree®Undefined Colombia
Feed additive for control of
Salmonella infection
in poultry
[57]
PhagePharm
(CHN) NuoAnSha Approved China Improve the breeding
environment [58]
Phagelab (CHI) Poultry
product Undefined Chile
Liquid food additive to
eliminate Salmonella
in broilers.
[50]
L.monocytogenes
Intralytix (USA)
ListPhage™ FDA
USA
L. monocytogenes in pet food [48]
C. perfringens
INT-401™ FDA, FSIS Against Poultry
C. perfringens [59]
PhagePharm
(CHN)
NuoAnSuoQing
Approved China
Necrotizing enteritis,
diarrhea, intestinal bleeding
caused by C. perfringens
[58]
staphylococcus
Delmont
Laboratories
(USA)
Staphage
Lysate(SPL)®FDA USA Staphylococcal skin infections
in dogs [47]
Yersinia ruckeri ACD Pharma
(NOR) CUSTUS®YRS FOT Norwegian Various bacteria in
aquaculture farms [60]
R. anatipestifer PhagePharm
(CHN) JiangYanQing Approved China
Decontamination of
R.anatipestifer in
aquaculture environments
[58]
Weisella ceti SciPhage (CO) Weissella Ceti
Phages Undefined Colombia control weissellosis in trout [61]
Microorganisms 2022,10, 1324 7 of 22
Table 2. Cont.
Target Bacteria Company Products Regulatory
Approval Certifications Application Ref.
Variety of
bacteria
CJ CheilJedang
Research
Institute of
Biotechnology
(KOR)
Biotector®S
Undefined
South Korea
Feed additive for poultry
and pigs against Salmonella,
C.perfringens, E. coli.
[35]
Phagelab (CHI) cattle product Chile
Food additive prevent
infectious diarrhea caused
by E. coli and Salmonella.
[50]
Proteon
Pharmaceuticals
(POL)
BAFADOR®EFSA Poland Fish feed additive against
Aeromonas and Pseudomonas [62]
PhagePharm
(CHN)
NuoAnQing
Approved China
Improve the breeding
environment [58]
YaLiNing
Varmsphage
(CHN) ChangShi Infections caused by E. coli
and Salmonella [63]
Cytophage
(CAN)
Poultry Feed
Additives Undefined Canada
Prevents the common
bacterial infections
in chickens [64]
swine bacte-
riophage
Against the common
bacterial infections in swine
Fixed-Phage (UK)
aquaPHIX™
Approved UK
Added to the feed as
a solvent
[37,49]
farmPHIX™ Feed additives
petPHIX™ Topical application of gels
and creams
Pathway
Intermediates
(KOR)
ProBe-Bac FDA South Korea ProBe-Bac SE for pigs;
ProBe-Bac PE for poulty [65]
Phagelux (CHN)
LUNIN
Approved China
for poultry diseases
[66]
LUZON for swine disease
LUMON for cattle disease
MicroMir (RUS)
Vetagin®
Approved Russia
Prevention of bacterial
endometritis, abscess and
myositis in dairy cows
[67]
Bronchophage
Control of common bacteria
associated with lower
respiratory tract disease
Phagovet Prevention of bacterial
diseases in broilers
5. Phages Products in Food Health
The use of antibiotics aggravates AMR in livestock and poultry products and the high
morbidity and mortality caused by foodborne pathogens has been a global burden [
68
].
Contamination caused by foodborne pathogens can be transmitted from production lines
to humans, ultimately threatening human health. Phages are desirable for the biological
control of foodborne pathogens as an effective natural and ecological alternative [
69
]. There
are also increasing studies on the effectiveness of phages against foodborne pathogens. For
example, Mengzhe demonstrated that phage STP4-A with a wide host range is effective
Microorganisms 2022,10, 1324 8 of 22
against Salmonella as a food additive [
70
]. Vikram demonstrated that phage preparation can
effectively reduce the level and prevalence of E. coli O157:H7 in food [
71
]. As early as 1958,
the U.S. Food and Drug Administration (FDA) recognized phages and their derivatives
as GRAS (generally recognized as safe) through the Food Additives Amendment to the
Federal Food, Drug, and Cosmetic Act [
72
]. Phages are primarily used in three depart-
ments: primary production, biological preservation, and biological harmlessness in the
food industry to ensure food safety [
53
]. Phages used in primary food production can
prevent foodborne pathogens from entering the human body through the food chain, which
is an excellent pre-harvest strategy. Livestock and poultry products are contaminated with
pathogens in the production, processing, distribution, and consumption links. The applica-
tion of phage products in postharvest can effectively reduce the presence of pathogens on
carcasses, packaging, and RTE poultry products [
68
]. The benefit of phages for postharvest
poultry processing is that they do not affect the quality senses and nutritional value of
food [48].
Phage products are currently used with high safety to eliminate pathogens in animal
food (meat products and dairy products) and plant food (fruits and vegetables). The FDA
has granted phage products GRAS approval, allowing them to be used in livestock and
poultry products. The use of phage products in food is also approved by health agencies in
Israel, Canada, China, Switzerland, Australia, New Zealand, and the European Union [
53
].
Since the FDA approved the first phage product, ListShield
™
, as a food preservative
in 2006, a significant number of phage products have emerged worldwide to combat
foodborne pathogens [
34
,
73
]. As of November 2021, 14 phage products have been used in
food processing, of which 11 have been approved by the FDA, including Intralytix (USA)
and Micreos (Utrecht, Netherlands). Table 3lists the commercial phage products used
to combat foodborne pathogens in detail. The statistics of approved commercial phage
products against foodborne pathogens revealed that Intralytix (USA) has made remarkable
contributions to the field, with five products for marketing, and has gained Jewish cleansing
and halal certification. Figure 2B reveals that commercial phage products primarily compete
with E. coli,Salmonella, and L.monocytogenes, which seriously threaten human health. It
is worth noting that Campylobacter is the most commonly reported foodborne pathogen,
but there are no commercial Campylobacter phage products. A recent project (C-SNiper)
directed by the Spanish Technology Center (AZTI) developed a prototype phage product
for Campylobacter that is expected to be globally commercialized in 2022 [74].
Table 3. Commercial phage products fighting foodborne pathogens in food.
Target Bacteria Company Phage
Products
Regulatory
Approval Certifications Application Ref.
E. coli
Intralytix (USA) EcoShield
PX™
FDA
Canada; Israel;
USA
Eliminate E. coli O157:H7
contamination prior to grinding
or packaging
[71,75]
Micreos (NED) PhageGuard
E™ USA
E. coli O157 on beef carcasses,
primals, subs and trimmings. [76]
FINK TEC GmbH
(GER)
Secure Shield
E1
Used in beef products, turkey
and other foods [48]
Salmonella
Intralytix (USA) SalmoFresh™ USA, Canada,
Israel
Food additives for poultry, fish,
shellfish, fruits and vegetables. [77]
Micreos (NED) PhageGuard
S™
Canada; Israel;
Halal;
OMRI; SKAL
In spray or dipping form for
poultry, meat. [78]
Phagelux (CHN) SalmoPro®Canada, China As an antibacterial processing
aid in food. [56]
Arm and Hammer
Animal & Food
Production (USA)
Finalyse
™
SAL
Undefined USA For Salmonella in
poultry products. [79]
Microorganisms 2022,10, 1324 9 of 22
Table 3. Cont.
L.
monocytogenes
Intralytix (USA)
ListShield™
FDA
USA
Food additives for poultry, fish,
shellfish, fruits and vegetables.
[80]
Listex™ [81]
Micreos (NED) PhageGuard
Listex™
Swiss; Israel;
Halal; Canada;
Kosher
OMRI; SKAL
In spray or dipping form for
poultry, meat. [82,83]
Campylobacter Intralytix (USA)
Compyshield
™
USA
Food additives for raw red meat
[84]
Shigella Intralytix (USA) ShigaShield™ Removal of Shigella from meat
and vegetables [53,85]
Variety of
bacteria
Brimrose Technology
Corporation (USA) EnkoPhagum Approved Salmonella, Shigella, E. coli,
Staphylococcus in meat products. [53]
Fixed-Phage (UK) safePHIX™ Undefined UK Against bacteria in the food
cold chain [38]
6. Phage Products in Human Health
The application of phages to treat human diseases dates back to the successful injection
of phage preparations in France in 1921 to treat five children with dysentery caused by the
Shigella infection [
24
]. Belgian researchers published the first paper in the same year on
the successful use of phages to treat furuncles and carbuncles of human skin [
9
]. Initially,
the French company L’Oréal sold five phage preparations for the treatment of bacterial
infections, Antipiol (Deutsch, Germany) produced Enterofagos, and EliLily (Indianapolis,
IN, USA) first sold “Staphylofel” phage preparations for the treatment of streptococci and E.
coli [
86
]. D’Herelle and Eliava first used phages to control cholera in India in 1931 and found
no side effects following treatment [
87
]. During World War II, phages were also applied by
both Soviet and German armies to treat wound infections, with the German army using
Shigella phage preparation “Polyfagin” by Behringwerke Leverkusen to treat and prevent
dysentery in soldiers [
88
]. In the late 1930s, however, the Committee on Pharmacy and
Chemistry of the American Medical Association stated that the efficacy of phage therapy
was unclear and further research was needed. Together with the discovery of penicillin,
which led to the successful introduction and widespread use of antibiotics, interest in phage
therapy has diminished, with only the Soviet Union and some countries in Eastern Europe
still investigating it [14].
Common infections or minor injuries may be fatal with the increasing threat of AMR
to humans. Researchers found great potential for phage therapy and phage therapies
are increasingly being used for human bacterial diseases. In 2000, clinical human trials
using phage therapy as a potential antibiotic alternative officially began in the United
States, and phase I clinical data was first published in 2009. Clinical trials have revealed
that phage cocktails against E. coli, S. aureus, and P. aeruginosa are safe for the treatment of
wounds [
89
]. In 2013, the European Commission supported the large multinational phage
therapy multicenter clinical research program “Phagoburn”, which treated 27 patients
infected with P. aeruginosa burn wounds with phage therapy in France, Belgium, and
Switzerland [
90
]. Despite the intended purpose not being achieved, this is the first time that
three national regulatory agencies reached a consensus about phage cocktails for human
therapy. At present, there are five phage therapy institutions worldwide, which are: Eliava
Phage Therapy Center (Tbilisi, Georgia), Phage Therapy Center (Tbilisi, Georgia), Center
for Innovative Phage Applications and Therapeutics (West Philadelphia, PA, USA), Phage
International (San Ramon, CA, USA), and Phage Therapy Unit (Wrocław, Poland). Eliava
Phage Therapy, founded in 1923, was the first institution to focus on phage therapy and
has marketed phage cocktail products targeting specific pathogenic bacteria to treat human
bacterial infections [91].
It has been confirmed that phage therapy has a lethal impact on a range of bacteria,
which has contributed to an increase in phage therapy research and development for
Microorganisms 2022,10, 1324 10 of 22
human diseases by multiple institutions around the world. However, no phage products
have been approved for human use in the European Union or the United States. The FDA
has merely opened up the regulatory pathway for phages to provide a green channel for
phage products for clinical use in emergencies. Phage therapies are approved for use in
emergency treatment plans in the European Union, Australia, France, and Belgium [
92
].
Detailed information on phage products currently approved and in preclinical studies
worldwide is provided in Table 4. Figure 2C reveals that phages in preclinical products are
almost exclusively targeted at MDR bacteria, especially “ESKPAEE” pathogens, including
E. faecium,S. aureus,K. pneumoniae,E. coli, and others. It can treat the infections caused
by these bacteria at different sites, including bone and joint infections (IOA), diabetic foot
ulcers (UPD), and MARS caused by S. aureus [
93
,
94
]. It can treat the fibrosis and burn
infections caused by P. aeruginosa [
95
]. It can treat urinary tract infections and IBD caused
by E.coli and K. pneumonia [
96
,
97
]. Among the preclinical phage products, the cocktail
products composed of 4–8 phage mixtures account for 60% of the total.
Table 4. Phage products for human health.
Mono Component
Target
Bacteria Company Product Regulatory
Approval
Route of
Administration Application Ref.
E. coli
Intralytix
(USA) EcoActive™ FDA approved
IND, Phase 1/2a oral Targeting
adherent-invasive E. coli [98]
Pherecydes
Pharma
(FRA)
PhagUTI Phase I/II Undefined Treating E. coli Urinary
Tract Infections [99]
Phico
Therapeutics
(UK)
SASPject PT5 Uundefined Intravenous
injection
Fights diseases caused by
E. coli [100]
P. aeruginosa
Microgen
(RUS)
Bacteriophage
P. aeruginosa
Russian
Federation
national standard
certification
Oral intrarectal,
or Intracavitary
injection
Treatment and
prevention of diseases
caused by P. aeruginosa
[101]
Armata
(USA)
AP-PA02;
AP-PA03
FDA approved
IND, Phase 1b/2 Inhalation
Treatment of respiratory
tract infections
caused by P. aeruginosa,
especially in patients
with CF
[95]
BiomX (USA) BX004 Preclinical Oral [102]
Phagelux
(CHN) PGX0100 FDA approved
IND, preclinical Transdermal Spray and gel for
burn care [103]
Phico
Therapeutics
(UK)
SASPject PT3 undefined Undefined Against P. aeruginosa
infection [104]
Pherecydes
Pharma
(FRA)
Pneumo Phage
Phase I/II clinical
trials are
expected to start
in 2023
Inhalation
Treatment of acute
P. aeruginosa respiratory
tract infection
[99]
Microorganisms 2022,10, 1324 11 of 22
Table 4. Cont.
Mono Component
Target
Bacteria Company Product Regulatory
Approval
Route of
Administration Application Ref.
S. aureus
Microgen
(RUS)
Staphylococcal
bacteriophage
Russian
Federation
national standard
certification
Inhalation
Treatment of
Suppurative
Inflammation and
Intestinal Disorders
Caused by Staphylococci
[101]
Armata
(USA)
AP-SA01;
AP-SA02
FDA approved
IND,
Phase 1b/2
Intravenous
injection
Treatment of resistant
and refractory
Staphylococcus aureus
bacteremia and diabetic
foot ulcers
[93,94]
BiomX (USA) BX005 Preclinical stage
Transdermal
Atopic dermatitis caused
by S. aureus [102]
Phagelux
(CHN)
PL-01-SZ China NMPA
IND submission
expected in 2022
S.aureus lyase, a
hydrogel formulation for
the treatment of eczema
[103]
PL-06-FC
P.acnes and S.aureus lyase,
hydrogel for
acne treatment
iNtODEWorld
(KOR)
N-Rephasin®
SAL200 Phase II Intravenous
injection Effective against MRSA [105,106]
Pherecydes
Pharma
(FRA)
Phage Cocktail Phase I/II
Undefined
Fights bone and joint
infections (IOA) and
diabetic foot ulcers
(UPD) caused by
S.aureus.
[99]
Phico
Therapeutics
(UK)
SASPject PT1.2 Phase I
Engineered phages
deliver genes for
antimicrobial proteins
(SASPs) that rapidly kill
S. aureus
[107]
Staphylococcal
Eliava Bio
Preparation
(GEO)
Staphylococcal
Bacteriophage
Georgian
Approval
Oral or intrarectal
Prevention and
treatment of
postoperative
wound infections,
Staphylococcal infections
[108]
K.pneumoniae
BiomX (USA) BX003 Phase I Oral
Targeting K. pneumoniae
bacterial strains
present in the gut of IBD
and PSC patients
[102]
Shigella
Intralytix
(USA)
ShigActive™ FDA approved
IND,2021 Oral
Prevention of human
diseases caused by
Shigella infection
[109]
Enterococcus VRELysin™ Undefined Undefined
Colonization with
antibiotic-resistant
Enterococci and
associated bacteremia
[84]
C difficile AmpliPhi
(UK)
AmpliPhage-
004 Pre-phase 1 Undefined
Against C. difficile
(including highly
virulent RT027)
[104]
Microorganisms 2022,10, 1324 12 of 22
Table 4. Cont.
Mono Component
Target
Bacteria Company Product Regulatory
Approval
Route of
Administration Application Ref.
Streptococcal
Microgen(RUS)
Streptococcal
bacteriophage
Russian Federation
national standard
certification
Oral, topical and
intrarectal
Treatment diseases
caused by Streptococcus [101]
Gardnerella
spp
BioNTech
R&D(AUT) PM-477 Preclinical Undefined
Recurrent bacterial
vaginosis, synthetic
lysosomes
[110]
Fusobacterium
nucleatum BiomX (USA) engineered
phage Preclinical Intravenous
injection
Targeting Fusobacterium
nucleatum bacteria
present in the tumor
micro environment.
[111]
Combining targets against variety of bacteria
Dosage
Form Company Product Regulatory
approval
Route of
administration Application Ref
Phage spray
Biochimpharm
(GEO)
Phagyo®spray
Georgian
Approved
Topical
Treatment and
prophylaxis of bacterial
purulent–inflammatory
infections (multiple
microorganims)
[112]
Phage tablet
Septaphage
®
table
Oral
Phage
cocktail
Septaphage®
Phagyo®
PhageStaph
Phage
capsule Travelphag™ For bacterial infections,
indigestion
Phage
cocktail
Microgen
(RUS)
Salmonella
groups A,B,C,D,
bacteriophage
Russian
Federation
national standard
certification
Oral, intrarectal
Treatment and
Prevention of Diseases
Caused by Salmonella
[101]
E.coli-Proteus
bacteriophage
Oral, topical and
intrarectal
Treatment and prevention
of purulent inflammatory
and enteric diseases,
dysbacteriosis caused by
bacteria Proteus and
enterotoxigenic E.coli
Klebsiella
purified
polyvalent
bacteriophage
Specific lysis of K.
pneumoniae,K. odorifera,
K. rhinosclerosis.
Intesti-
bacteriophage
Treatment and
prevention of bacillary
dysentery
Sextaphage ®
Polyvalent
Pyobacterio-
phage
Treatment and prevention
of purulent inflammation
and intestinal diseases
Complex
Pyobacteriophage
Specific lysis of
Staphylococcus,
Streptococcus, Enterococcus,
Proteus, K.pneumoniae,
P.aeruginosa and E. coli.
Dysentery
polyvalent
bacteriophage
Oral and
intrarectal
Specific lysis of the
bacillary dysentery
pathogen
Microorganisms 2022,10, 1324 13 of 22
Table 4. Cont.
Mono Component
Target
Bacteria Company Product Regulatory
Approval
Route of
Administration Application Ref.
Phage
cocktail
Eliava Bio
Preparation
(GEO)
Pyo-Phage
Georgian
Approved
Oral, intrarectal,
or intracavitary
injection Treatment and
prevention of bacterial
purulent inflammation
and intestinal infections.
[113]
Fersisi-Phage
Intesti-Phage
Oral or intrarectal
SES-phage Rectal, or intra-
cavitary injection
ENKO-Phage Oral
Phage spray
Aziya
Immuno-
preparat
(UZ)
Bacteriophage
Staphylococcus
spray MediPhag
Marketed
Topical(spray)
A mix of sterile lysate
phages against S. aureus
[114]
Phage
cocktail
Bacteriophage
Staphylococcus
liquid MediPhag
Oral
Bacteriophage
Salmonella
polyvalent
MediPhag
Treatment and
prevention of multiple
serotypes of Salmonella
Bacteriophage
Dysenteric
Polyvalent
MediPhag
A mix of sterile lysate
phages against Shigella
GastroFag
polyvalent
MediPhag
Fight enteric diseases such
as Salmonella, Proteus,
S.aureus, P.aeruginosa, E.coli
Phage
capsule
Bacteriophage
dysenteric
polyvalent
“MediPhag”
A white gelatin capsule
containing
lyophilizied dried
bacteriophage capsules
against Shigella
phage tablet
MB Pharma
(CZ) LYZODOL®Marketed Oral
Against S.aureus,
K.pneumoniae,Lelliottia
amnigena,
Propionibacterium acnes
causing respiratory
infections.
[115]
Phage gel MicroMir
(RUS)
Phagodent
Marketed Topical
Contains 72 phage
complexes to normalize
oral microflora
[76]
Phagoderm
Skin gel containing 64
phages to prevent bacterial
infection of the skin.
Phagogyn
Gel containing 74 phages
that prevent bacterial
diseases of the
reproductive system.
Otophagus
Gel containing 69 phages
that prevent bacterial
and suppurative
inflammation of the ear,
nose and throat
Microorganisms 2022,10, 1324 14 of 22
Table 4. Cont.
Mono Component
Target
Bacteria Company Product Regulatory
Approval
Route of
Administration Application Ref.
Phage
cocktail
Phagex (UKR)
Pyofag®
Marketed Oral and topical
Treatment of pathogenic
factors in purulent
inflammation and
intestinal diseases
caused by Streptococcus
pyogenes, S.aureus, E.coli,
P.aeruginosa, Proteus
vulgaris, Proteus mirabilis [116]
Intestifag®
polyvalent
bacteriophage
Fights intestinal diseases
caused by Shigella,
Salmonella, E. coli, P.
aeruginosa, Enterococcus
faecalis, S. aureus
Phico
Therapeutics
(UK)
SASPject PT4 Undefined Intravenous
injection
Treatment and
prevention of diseases
caused by K.
pneumoniae and E.coli
[100]
Phagelux
(CHN) BACTELIDE™
FDA approved
IND,
preclinical
Transdermal Patches and sprays for
pressure ulcers [103]
Fixed-Phage
(UK) mediPHIX™ Undefined Undefined Effective against a
variety of bacteria [38]
Adaptive
Phage
Therapeutics
(USA)
PhageBank
FDA approved
IND,
Phase 1/2
Intravenous
injection
Treat Diabetic Foot
Osteomyelitis, Prosthetic
Joint Infection, Chronic
Recurrent UTI,
Ophthalmic Infection,
Cystic Fibrosis-related
Lung Infection
[117]
Locus
Biosciences
(KOR)
crPhage™ Phase 1b Injection
Combined with
CRISPR-Cas3 to enhance
bactericidal efficacy
against various bacterial
diseases such as IBD
and UTI
[118]
Ellis Day Skin
Science (USA)
Balancing
Phage
Serum Marketed
Transdermal
Eliminate bacteria
associated with
blemishes
and acne to balance the
skin microbiome
[119]
Hydrating
Phage
Serum
PHYLA
(USA)
Phortify
Probiotic
Serum
Marketed
A probiotic serum that
targets and neutralizes
acne-causing bacteria
[120]
SciPhage
(CO) AcneFree Undefined Fights acne-targeting
bacteria [61]
Phage cocktails can increase the host range and avoid targeting a specific pathogen.
In addition, rapid identification of bacterial pathogens is a time-consuming and laborious
process before individualized treatment with phages [
121
]. Notably, phage cocktails are
still targets for treating bacterial diseases caused by “ESKPAEE” pathogens. Figure 2D
Microorganisms 2022,10, 1324 15 of 22
reveals that phage cocktail products that have been marketed in Russia and Georgia are also
basically liquid phage cocktails, with only a few gels, capsules, and tablets available. Due to
the effective identification of phages in the reticuloendothelial system, the half-life of phages
in humans is usually relatively short [
122
]. Figure 2E reveals that the route of administration
has a significant impact on the efficacy of phage absorption into the human body. Currently,
the administration routes of phage products in preclinical studies mainly include oral,
topical, transdermal, inhalation, and intrarectal administration. There are various routes of
administration for phage products, with oral administration accounting for 35% of the total
and remaining the most prevalent, followed by topical and intrarectal administration. Oral
administration is effective in delivering phages to the gastrointestinal tract but it is the least
effective route for systemic penetration. The most effective mode of delivery is an injection,
which may deliver phages to practically all organs and tissues in minutes. Therefore, the
efficacy of phage therapy is determined by the route of administration.
Endolysin and virosome-associated lysozyme (VAL), which are phage-derived
peptidoglycan-degrading enzymes, are also bactericidal. Endolysins are enzymes used
by phages to lyse the bacterial cell wall at the end of the replication cycle, while the VAL
is responsible for the injection of genetic material into infected cells for peptidoglycan
degradation [
18
,
123
]. Many studies on the antibacterial effect of endolysin are currently
being implemented in human medicine clinics. Endolysin is also one of the alternatives to
antibiotics. It has the advantages of killing the host quickly, host specificity, preservation of
the normal microbial community, reduction of AMR risk, and efficiency against multidrug-
resistant bacteria and biofilms when compared to antibiotics [
124
]. The benefits of endolysin
therapy have attracted the attention of researchers and pharmaceutical companies to its
commercial potential and several commercial products based on endolysin have now
been developed. The first human endolysin product developed by Micreos, Staphefekt
SA. 100, specifically for the treatment of chronic S. aureus associated skin diseases, has
been marketed. All three clinical patients had a positive therapeutic effect and did not
develop resistance [
125
]. Artilysin has developed an Artilysin
®
product line(Lysando AG,
Regensburg, Germany) that is effective against resistant P. aeruginosa and A. baumannii in
various forms including spray, nebulizer, solution, lyophilization, gel, and coating [
126
].
Rephasin®SAL200 (Intron Biotechnology, Seongnam, Korea) is now in phase II of human
clinical trials [
105
,
127
]. ContraFect has developed a novel direct lysing agent called Amurin
peptide, which is effective against numerous Gram-negative pathogens. The other is
a lysin-based direct lytic agent, containing Exebacase CF301, which is effective against
S. aureus, including MARS, and is the first phage lytic enzyme to enter human clinical
trials in the United States [
128
,
129
]. Criteria used for the preclinical analysis of small
molecule antibiotics may be more readily translated into the preclinical assessment of phage
lytic enzymes than phages, so clinical evaluation of phage lytic enzymes is progressing
significantly faster [130].
7. Advantages and Disadvantages of Phage Therapy
Compared to antibiotics, phages are characterized by host specificity, which means
only lysing the host bacterial cell wall without destroying the microbiota [
62
]. There is a
process of adaptation versus counter-adaptation in the coevolution of phages and bacterial
hosts and the risk of developing resistance is low [
40
]. For example, coevolutionary phage
training can delay the evolution of phage resistance. Researchers conducted coevolution
experiments using E. coli and untrained or trained phages to assess the potential of phage-
training treatments and found that trained phages were able to inhibit host bacteria for a
longer period of time [
131
]. The coevolution of phages with host bacteria has also driven
bacteria to evolve a variety of highly specific phage defense mechanisms. For example,
mutations in phage receptors, the R-M system, the DISARM system, the superinfection
exclusions (SIEs) system, the abortive infection (Abi) system, and the adaptive immune
system CRISPR-Cas all make phages resistant [
132
,
133
]. Hussain studied the evolutionary
trajectory of resistance in wild-type phages, which showed that the rapid evolution of
Microorganisms 2022,10, 1324 16 of 22
mobile phage defense elements (PDEs) drove bacterial resistance to phages [
134
]. Studies
have shown an evolutionary trade-off between phage and antibiotic resistance, with bacteria
sometimes showing increased susceptibility to antibiotics when phage resistance evolves.
Barber studies have demonstrated that efflux pumps play a dual role in antibiotic resistance
and phage sensitivity, and when phage resistance leads to the loss of bacterial capsules,
they will subsequently become sensitive to antibiotics [
135
]. However, when Burmeister
studied E. coli phages, it was found that bacterial interaction with phages may depend on
efflux pump protein TolC and structural barrier molecule lipopolysaccharide (LPS), and
when these two mutants were constructed, some phage resistance mutations conferred an
increase in antibiotic resistance [
136
]. Therefore, there are not only synergistic effects but
also antagonistic effects between phages and antibiotics, and their intrinsic mechanisms
of action remain to be further studied. In addition, phages replicate only in the target
bacteria at the site of infection and treatment causes fewer adverse effects and is safer. Oral
phage preparations are generally harmless and researchers have found the presence of
adverse effects associated with phage therapy when assessing animal and clinical phage
therapy safety and toxicity, but with a small probability of events [
137
]. Finally, phages are
widespread in the environment and provide an inexhaustible resource. It only takes days
to weeks to produce a new natural phage preparation, and if a phage develops resistance,
phages that use other new receptors can also be quickly found. Screening for a new natural
phage preparation takes a few days, and phages using other new receptors can also be
quickly found if the phage develops resistance.
Despite the favorable results of various studies on phage applications, phage therapy
still has certain shortcomings and unknowns. First, there are still some potential risks
associated with the application of phage therapy, which are largely observational with
existing phage therapies or performed in small non-randomized trials, where side effects
may be underestimated. Second, the route of administration, frequency of administration,
dose, phage resistance, pharmacokinetic and pharmacodynamic characteristics of phages,
and the mechanism of phage entry into eukaryotic cells and the immune system need to
continue to be studied in depth [40,138]. Third, legal regulation is a significant obstacle to
the implementation of phage therapy and regulatory authorities classify phages as biologi-
cal substances, which differs from the approval and production of antibiotics, making it
difficult to use phage therapy. European legislators have been advocating for a regulatory
framework specifically targeting individualized phage preparations but they have been
strongly resisted [
139
]. Fourth, considering phages are natural entities, they entrap pharma-
ceutical companies in intellectual property issues [
121
]. Fifth, animal prophylactic phage
products do not remove phages immediately after use and may lead to phage mutation
and the cultivation of phage mutants. This problem also needs to be solved by using the
regular rotation of phages and continuous detection, such as antibiotics [
35
]. Sixth, phages
can transfer bacterial resistance genes and even contain toxic genes, implying that, as much
as possible, the selection of lytic phages ensures that therapeutic phage products must be
deeply purified and must remove endotoxins during processing [
140
]. Seventh, when the
scope of phage application expands, including antibiotic substitutes, carrier delivery drugs,
vaccines, and phage display technology, the demand for large-scale production of phage
increases. The Phage on Tap (PoT) protocol has been studied for the rapid formulation
of high titer phage formulations and a systematic procedure has also been developed for
the isolation, up-culture, concentration, and purification of phages for pharmaceutical
use [
141
]. The procedure can combine modified classical techniques, modern membrane
filtration processes, and no organic solvents in 16 to 21 days, producing an average of
23 mL
of 10
11
PFU/mL phage [
142
]. Despite the enormous efforts of researchers for phage
technology, there remain challenges for the production and expansion of wild-type phages
for biological control. Finally, doctors and the public at large are unaware of the use of
phages to treat diseases and the public believes that viruses are exclusively harmful to the
human body, not realizing that they may also be beneficial [143].
Microorganisms 2022,10, 1324 17 of 22
8. Conclusions and Prospects
Antibiotic resistance poses a threat to global health. Russia approved the addition
of phages to the official pharmacopoeia in 2016. The European Pharmacopoeia included
“phage therapeutic active ingredients and pharmaceutical products for human and veteri-
nary use” in 2021. To ensure its safety and effectiveness, pharmaceutical authorities such as
the FDA and EMA require that any modern phage therapy product meet GMP standards,
which poses challenges [
144
]. According to the statistics, Figure 3C reveals that 20 countries
began to develop phages and had phage products approved for use. According to the
analysis of 123 products in 20 nations, Figure 3A reveals that 53% of the products were
used for human health, and Russia, Georgia, and the United States have rich experience in
phage therapy for human diseases. Figure 3B reveals that the FDA has approved twenty
phage products for animals and food, but only seven investigational new drugs (INDs) for
humans. Except for Russia and Georgia, which have focused on phage therapy for human
diseases and have sold many phage products, phage product research and development
in other nations remains to be further developed. Overall, phage products in the United
States are rapidly developing and the FDA has also approved several products. In the
future, as a novel alternative therapy under the “one health” approach, phage research and
development will continue to focus on making products that are environmentally friendly,
safe, and successful in combating AMR.
Microorganisms2022,10,xFORPEERREVIEW15of20
Figure3.Totalphageproductsaredistributedandapproved.(A)Theproportionofphageprod‐
uctsinplants,animals,food,andhumans.(B)Thenumberofphageproductsthathavebeenap‐
provedbytheFDAandareinclinicalresearchandmarketedforhumanuse.(C)Worldwidedis‐
tributionofthenumberofphageproducts.
AuthorContributions:Y.H.collecteddataandwrotethefirstdraft.W.W.andZ.Z.contributedto
thedatacollectionofthemanuscript.Y.G.,A.H.andJ.W.madesuggestionsforrevisionofthe
manuscript.H.H.providessupervision,guidanceandfinancialsupport.Allauthorshaveread
andagreedtothepublishedversionofthemanuscript.
Figure 3.
Total phage products are distributed and approved. (
A
) The proportion of phage products
in plants, animals, food, and humans. (
B
) The number of phage products that have been approved by
the FDA and are in clinical research and marketed for human use. (
C
) Worldwide distribution of the
number of phage products.
Microorganisms 2022,10, 1324 18 of 22
Author Contributions:
Y.H. collected data and wrote the first draft. W.W. and Z.Z. contributed to the
data collection of the manuscript. Y.G., A.H. and J.W. made suggestions for revision of the manuscript.
H.H. provides supervision, guidance and financial support. All authors have read and agreed to the
published version of the manuscript.
Funding:
This work was funded by grants from National Key Research and Development Program
(2021YFD1800600), National Natural Science Foundation of China (32172914), Fundamental Research
Funds for the Central Universities (2662022DKYJC005).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments: We thank the editors and peer reviewers for reading this manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
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