1 Background
Antibiotic resistance caused by the emergence of super bacteria,
poses a major threat to human health and has a significant impact on global economy and security.
In 2015, the World Health Organization launched the Global Antimicrobial Resistance Surveillance System
(GLASS).
In the past four years,
91 countries and regions have joined GLASS. On May 26,
who released the Global Antimicrobial Resistance Surveillance System (GLASS) report -
early implementation 2020, which collected 2365 data from 66 countries in 2019 Sample
data from 972 infected people showed that antibiotics often used to treat common bacterial
infections have a disturbing high rate of drug resistance, and more and more bacterial infections
have consequently developed drug resistance. In the case of ciprofloxacin, an antibiotic used to treat
urinary tract infections, the drug resistance rate is between 8.4% and 92.9% in more than 30 countries.
In addition, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae and a series of other
bacteria have
long been drug-resistant. The report points out that these data are very important to solve the problem of
drug resistance.
If more and more bacteria develop drug resistance, we will have to face the terrible situation of no drug
available in the future.
A report prepared by economist Jim O'Neill was also quoted in the 2016 who bulletin. It is estimated that
about 700, 000 deaths worldwide
2016 can be attributed to antimicrobial drug resistance. 35 years from now, the death toll will rise to
10 million a year, more than
the current number of cancer deaths. The report predicts that if no measures are taken to reverse this
trend, the total cumulative loss will
be as high as $100 trillion by 2050. In reiterating the impact of antibiotic resistance on public health,
the Secretary General of the United
Nations pointed out in May 2019 that antibiotic resistance is a global threat to health, livelihoods, the
environment and the achievement of
sustainable development goals. In 2019, an article was published in the international environmental Journal,
with the tile as "Antibiotics
bioremediation: perspectives on its ecotoxicity and resistance". It was mentioned in the literature that
antibiotics are used as
growth promoters in animal husbandry, such as cattle, pigs and poultry, to improve feeding efficiency.
Although they were banned
in the EU in 2006, they are still used in some developing countries. Their release into the environment and
natural ecosystems may
interfere with critical bacterial cycling processes,
which are critical for water balance or agricultural balance and animal production. Antibiotics have been
reported in hospital wastewater, soil, groundwater and drinking water.
In China, the detection rate of carbapenem resistant Acinetobacter baumannii was as high as 55.4%,
and the detection rate of imipenem resistant Acinetobacter baumannii (IMP-R-ABA) was also as high as
55.4% The detection rate of cefotaxime resistant Escherichia coli (CTX-R-ECO) was 52.6%. The
detection
rate of other common clinical drug-resistant bacteria also showed a slow upward trend. If bacterial
resistance
is not addressed, one million people will die early each year. Large scale bacterial diseases often
occur in animal breading industry. Streptococcus suis can harm 1-5% of the pig population, and the
sick pigs may die and suffer
from meningitis. Cow mastitis is caused by pathogenic bacteria such as Staphylococcus aureus, and the
prevalence rate
in dairy cows is as high as 30-70%, which seriously endangers the development of dairy industry. With the
abuse of
antibiotics, the main pathogenic bacteria in livestock production gradually produce drug resistance, which
makes the prevention
and control of bacterial diseases more difficult. According to statistics, in 2013, 162 thousand tons of
antibiotics were used in
China mainland, accounting for about half of the world's total consumption,
of which 52% were veterinary (84.24 thousand tons), 48% were for human use, and over 50 thousand tons of
antibiotics
were discharged into the water and soil environment. This would pose a serious threat to human health,
animal breeding industry, food safety, and environmental pollution.
2 What Can We do?
Antibiotics have been widely used in the past few decades,
which has greatly increased the antibiotic resistance of bacteria,
and caused multi-drug resistance or super bacteria while it has a
great impact on the environment, economy and society. Thus, we need
new therapy methodologies and programs to control bacterial infection
while phage therapy is one of the most promising technologies for the
treatment of bacterial infections. However, phage therapy has not been
widely used due to various limitations, and even some professional
infection doctors are not familiar with it. We plan to solve these
problems by manipulating the phage genome. Here we are working on a
platform to effectively reboot the yeast phage. Our platform will
simplify the manufacturing process of custom-made phages, which will be
safer and more controllable for treatment. In addition,
we will promote phage therapy and reduce the use of antibiotics by developing huge-sized lytic phage genome
manipulation platform.
3 Overview
While completing our project design and experimental parts,
our ultimate goal is to put scientific research results into
practice. We’ve been thinking, what is the significance of our project?
What problems can be solved? What kind of value can be created? As a team, we always
believe that it is vital to have a lasting impact on others and society through our project.
This is why we try our best to understand the needs of stakeholders in the process of promoting
the project, and we will consult the implementation of antibiotics feedback from experts, doctors,
manufacturers and the public to promote our project, while ensuring that our project is biosafe,
responsible, feasible and sustainable. In the following content, you will learn about our progress,
thinking process and how we integrate expert opinions. We divide the project into four phases.
In different phases, the team focuses on various issues related to bacteriophages,
which affect society and the progress of the project. We believe that our solutions can bring great benefits
to the community.
4 The First Stage
At this stage, we interviewed as many stakeholders as possible regarding the status of bacterial resistance
in China, the problems and challenges faced by the use of antibiotics, etc., to understand the situation of
super bacterial infections and the impact of antibiotics on the economy, society and environment. In this
way, we can understand our problems from multiple angles and think about how our projects can better affect
society.
Medical Staff
Interview reasons:
Medical staff are the first people to be able to contact patients with bacterial infections.
They have a certain understanding of super bacterial infections,
treatment options for bacterial infections, the use of antibiotics and their side effects.
Through interviews with our local senior doctors, we hope to understand the current status of bacterial
infections in China,
the frequency of super bacteria, and the shortcomings and deficiencies of existing treatment methods for
bacterial infections.
Interview contents:
Dr. Lv Mingyi，female, the chief of MICU, at of Zhongshan Hospital affiliated to Dalian University, graduated
from Dalian Medical University. She has been engaged in critical care medicine for more than 10 years and
has accumulated rich clinical experience. She is expert in diagnosis and treatment of critical care diseases
in critical care medicine, and has published several SCI and core journal articles. She told us that
patients with bacterial infection would be received in the infectious department, surgery department,
respiratory department and ICU of the hospital, and some departments reached about 10% of the total number
of patients. In the ICU ward, the death rate due to drug-resistant bacteria infection is relatively high. It
can even be convinced that the emergence of drug-resistant bacteria will cause the death of patients. At
present, the first choice for the treatment of bacterial infections is broad-spectrum antibiotics, together
with some auxiliary drugs. However, after the use of broad-spectrum antibiotics, bacterial resistance may
gradually develop. In addition, antibiotics may lose their effect of anti-bacteria, and side effects such as
damage to liver cells and blood system, may occur simultaneously. The consumption of wealth in antibiotics
therapy is, sometimes, too giant. Some patients will choose to quit the treatment and finally die just
because they cannot afford expensive antibiotics. At present, there is no cure for multi-drug resistant
bacterial infections and doctors also hope to solve this problem as soon as possible.
2) Dr. Song Xiaoping, female, the chief physician and director of respiratory Department, at Zhongshan
Hospital affiliated to
Dalian University,
Master of Medicine, has been engaged in the major of respiratory diseases for 23 years.
She is good at the diagnosis of respiratory infectious diseases and chronic
obstructive pulmonary diseases, and has in-depth research on some difficult diseases in the department of
respiratory medicine.
From Dr. Song, we learned that bacterial infections occur in the three major pipelines of the human body:
respiratory tract,
digestive tract, and urogenital tract. And more than 90% patients are treated with broad-spectrum
antibiotics,
while other methodologies are rarely applied. Doctors hope that antibiotics with narrow spectrum will be
developed,
but due to the rapid mutation of bacteria, the current research and development speed of antibiotics cannot
meet the
market demand. In addition, multi-drug-resistant bacteria, such as some strains of Staphylococcus
aureus, Klebsiella
pneumoniae and Pseudomonas aeruginosa, cannot be destroyed
by the antibiotics currently used, so a novel solution to struggle against bacterial infections is urgently
called for.
What did we get from the interview with doctors?
From the interviews with local doctors, we learned that bacterial infection is relatively common and
antibiotics mainly used to treat bacterial infection. The disadvantages of using antibiotics to treat
bacterial infection include: (i) the development of novel antibiotics is slow; (ii) antibiotics cause
drug-resistance easily, (iii)
usual antibiotics is relatively expensive , (iv) low specificity, and (v) at a loss as to how to deal with
super bacteria.
In terms of medical treatment, there is an urgent need for a substitute to replace antibiotic therapy.
Related Staff in animal breeding Industry
Interview reasons:
China is a large country in the production and consumption of animal-derived food.
In order to improve the breeding efficiency, farmers mostly adopt intensive breeding mode,
which has the shortcomings of causing high incidence of epidemic diseases.
In order to solve the problem of animals dying from disease, farmers usually
mix a large number of antibiotics and other drugs in the feed. As a result, drug
resistance due to continuous feeding or abuse of antibiotics continues to grow, making
disease control increasingly difficult. We hope to understand the role of antibiotics
in farmed animals and the situation of infections in farmed animals through interviews with farmers and
others.
Interview contents:
1) We followed the college's practice team of "Green Agriculture and Rural Revitalization" to visit nearly
fifty farmers from six province to understand the status of antibiotics usage in animal breeding industry.
Hao Xiaobao, a farmer in Weishi
County, Henan Province, told us that diarrhea in piglets alone can lead to the death
of more than 50 piglets every year. Ren Junhai, a farmer in Fuping County, Hebei Province told us
it has not been effectively solved all year round that the high incidence of piglet
diarrhea in autumn and winter every year, with a mortality rate as high as 10%. In
Tongwei, Gansu Province, nearly half of the animals suffered from diarrhea
and dysentery in a village Breeding Plant, with little effect of antibiotic treatment. Because animals are
easy to
relapse after dosing, difficult to be radically cured, and suffer from great side effects.
2) Cong Cong, PhD student, School of Biological Engineering, Dalian University of Technology
Dr. Cong Cong has done a lot of research on the use of antibiotics in animal husbandry and phage therapy.
She told us that most farmers in our country usually mix a lot of antibiotics and other chemicals in their
feed to solve the disease problem, but antibiotics The unreasonable (long-term, low-doses) use
of antimicrobial bacteria has caused a large number of drug resistance of pathogens, increased
the difficulty of disease prevention and control. This not only directly leads to a large number
of drug residues in food and environment, pollutes soil, air and water, but also directly or
indirectly harms human health, sowing a huge crisis for the society. According to statistics,
about 24 million pigs die from diarrhea every year due to pathogenic microorganisms, causing about
12 billion economic losses. Since 2015, policies to restrict or stop antibiotics have been continuously
introduced. The Ministry of Agriculture and Rural Affairs of The People's Republic of China issued
Announcement No.
194 this year, which clearly stated that the production of feed antibiotics will be completely stopped
starting from
July 1, 2020. Therefore, bacteriophages, as a green, safe and efficient new
antibiotic substitute, have practical and reliable practical significance for the aquaculture industry in
China.
What did we get?
Due to the long-term use of antibiotics in animals, the emergence and
detection of drug-resistant bacteria is accelerated, resulting in the decline of
animal immunity. The situation of drug resistance of bacteria from animals is
increasingly severe, and the therapeutic effect of antibiotics is gradually reduced.
Though using large dose of antibiotics, livestock are still cannot survive from the
pandemic caused by bacteria, which has caused great pecuniary loss to local farmers.
In addition, due to the continuous introduction of national policies, the application of
feed antibiotics has been strictly restricted. Thus, the whole breeding industry is looking
forward to a safe and efficient substitute for antibiotics to ensure the safety of animal derived
food and improve the level of animal health. As we can see, phage has a broad prospect in the animal
breeding industry.
Antibiotic Manufacturers
Interview reasons:
Antibiotic manufacturers have a good understanding of the demand for antibiotics in medical treatment,
animal husbandry and other aspects. They can get feedback from consumers and users,
so that they have a certain understanding of the latest national policies on the use
of antibiotics, and have studied the development situation of the entire antibiotic industry.
We hope that by interviewing them, we can understand the current information on the use and
consumption of antibiotics, and measure the future application prospects of phages.
Interview contents:
Yangtze River Pharmaceutical Group is a drug R & D manufacturer, involving Chinese and Western medicine,
prescription drugs and other fields, which are applied in the treatment of anti-microbial, anti-tumor and
other diseases. Five of the Chinese herbal medicines have entered the European pharmacopoeia standard, and
won 20 international QC gold awards in 2015-2019, with a 2020 brand value of 50.595 billion RMB.
Dr. Liu Jinglong, who is one of the backbone scientists, at Institute of Pharmaceutical Research, Yangtze
River Pharmaceutical Group Co., Ltd. He told us that at present, most antibiotic manufacturers have started
the research on antibiotics against super bacteria with strong drug resistance. However, from a market
perspective, due to the existence of strong antibiotic resistance is a small number of patients, the
clinical demand for broad-spectrum antibiotics is still very large.
The Northeast Pharm has major business sectors such as chemical preparation, pharmaceutical engineering,
biomedicine, etc., mainly producing vitamin series drugs, anti infective drugs, antiviral drugs and other
excellent products. The leading products have passed the registration and quality audit of many countries
and regions such as the United States, Europe, Japan, Brazil, Russia, Poland, etc., exported to more than
100 countries and regions, and many products have passed the international high-end certification such as
EDQM, FDA, Japan's Ministry of health and welfare, BRC, HALAL, KOSHER, etc.
Mr. Liu, the head of the sales department of Northeast Pharma told us that although antibiotics can help
many people to avoid infections caused by bacteria and the market demand is still very large, but due to the
increase of antibiotic resistance year by year, many countries have issued the relevant policy of "limiting
resistance", and the use of antibiotics will gradually return to departments and hospitals. The factory has
also been hit to a certain extent. The international control of antibiotic application is becoming
increasingly strict, making patients and doctors more cautious about the use of antibiotics.
Shandong Lukang Pharmaceutical Co., Ltd. (hereafter referred to as Lukang Pharma), found in 1966 in Jining,
Shandong Province, is a state-owned large-scale comprehensive pharmaceutical enterprise, which was listed on
the main board in 1997 (Stock abbreviation: Lukang Pharma, Stock No.: 600789). Lukang Pharma mainly produces
raw materials and preparations for human use, animal and plant drugs, including more than 500 varieties of
anti-infective drugs, cardio cerebrovascular drugs, diabetes, amino acids and so on. Many of the API
products have passed the cGMP certification of EU and FDA certification of the United States. Lukang
Pharma's products are sold well all over the country and exported to more than 50 countries and regions in
Asia, Europe, Africa and America, with an annual export value of 150 million US dollars.
Mr. Yin, the director of Dalian Office of Lukang Pharma told us that the public has already had a certain
psychological resistance to antibiotics. Some patients are seriously inflamed but, however, they still
refuse to use antibiotics. They think that antibiotics will reduce the body's immunity and even cause some
else serious illness. However, as long as we use antibiotics rationally, the positive effects are still
obvious and little side effects would be caused. After listening to our introduction of bacteriophages, Mr.
Yin thought that phage therapy is very promising. Now the development of novel drugs is a race against time.
Because of bacterial mutants and drug resistance, the research of antibiotics has encountered a bottleneck.
If phage therapy can be developed and optimized rapidly, it would be finally used in clinical treatment and
the damage to human body can be minimized. In a word, it is a good technology and methodology.
What did we get?
China has issued relevant strict policies to limit the use of antibiotics at present while antibiotic
pharmaceutical factories have also been affected to a certain extent. People have become more cautious about
the use of antibiotics. Some of the public have a sense of resistance to antibiotics, that antibiotics will
harm human health. For drug-resistant bacteria, many antibiotics have become ineffective. Thus, phage
therapy is promoting to act as a substitute of antibiotics if the technique of phage therapy is developed.
Conclusion
Through interviews with doctors, veterinary, farmers, and antibiotic manufacturers, we learned that
bacterial infection often occurs with unfortunate impacts. However, antibiotics, as the main therapy
strategy, are gradually reduced or even completely ineffective when facing the multi-drug-assistant
bacteria. What’s worse, the use of veterinary antibiotics has caused great harm to the environment, ecology,
economy and society. In terms of policy, China has also issued a series of policies to limit the use of
antibiotics dealing with the urgent situation. Altogether, we can see that many industries such as medical
treatment, animal husbandry or even the whole society are looking forward to the emergence of green, safe
and efficient antibiotic substitutes.
5 The Second Stage: Explore the project and significance
In order to solve the problem of bacterial infection, we hope to find a safe and efficient approach to
reduce the use of antibiotics. At first, we summarized and compared the
certain antibiotic alternatives by consulting the literature. Among them, we regard phages as the most
potential antibiotic substitute due to its strong antibacterial specificity,
green and no residue. Phages has been certified by the U.S. FDA and belongs to the Gras (generally reclaimed
as safe) product. We believe it is of great practical significance to
develop and promote bacteriophage products to apply them to bacterial infection. For further understanding
of its characteristics, advantages and disadvantages, we visited
bacteriophage experts and clinical researchers to think about which problems should be targeted by us to
solve?
Literature Review
According to previously published articles, we found that previous researchers have put forward a lot of
feasible schemes to prevent the antimicrobial spreading and
antibiotics abusing. The most commonly used schemes are the followings:
1) Antimicrobial peptides:
Antimicrobial peptides have excellent performance on antimicrobial effect and barely cause antimicrobial
resistance. However, antimicrobial peptides are easily to be
degraded and their effect are unstable. And the high cost of chemical synthesis makes it nearly impossible
to be popularized.[1]
2) Chinese herbal extracts:
The natural Chinese herbal medicine has low side effects probabilities and is non-pollution. However, its
disadvantages are also apparent. Its development is
largely limited by its slow and instable efficacy.[2]
3) Vaccines:
Many available Vaccines have good performance in therapies. It only need to be stored and transported at 2 ~
8 ℃，which is relatively convenient. However, there are only few
feasible ways of inoculation, which are mainly done through subcutaneous or intramuscular injections. And in
most cases, vaccines take nearly 2 to 3 weeks to stimulate immune systems
to produce immunity. Therefore, it is not suitable for prophylactic immunization with emergency.
4) Phages:
Phages have high specificity and are expected to nearly have no effect on normal microflora. But the narrow
host spectrum of available phages makes it nearly impossible to be applied more widely.
On the basis of our investigations, we have found that following progresses has been made in the phage
therapy:
1) Applications of phages
Phage therapy has been tested in laboratory animals and achieved comprehensive success.[3]
2) Phage cocktails
Currently, the existing types of phage cocktails are: whole bacteriophage cocktails administered by
injection or oral administration. The results of animal experiments showed that the phage could be rapidly
distributed and reach the infected site. In addition, the higher the phage concentration is, the better the
effect it is. On the other hand, people try to purify the specific lyases of phages as a therapeutic
preparation. These chemical molecules are cytoderm lyases, working by binding specifically to glycosyl
groups in cell cytoderm. Animal experimental tests show that this method has a strong bactericidal effect.
Therefore, we are interested in phages and hoped to use phages as a feasible substitution to antibiotics. We
continue further literature research to better understand the advantages and disadvantages of phages. Then,
we started thinking about what we can do to promote phage therapy with synthetic biology methods.
Phage has following advantages:
1) High selectivity and specificity:
Phages have unique bactericidal mechanism with high specificity, so that the phages are not threatening to
other symbiotic bacteria in humans and animals’ bodies.
2) Low probability of drug resistance:
Compared with antibiotics, phages are able to degrade bacterial biofilms that is essential to confer its
hosts the resistance to antibiotics. Therefore, we make use
of phages to lyse biofilms of multi-drug-resistant bacteria. Besides, phages can evolve and keep abreast of
the evolution of bacterial resistance by amplifying in their corresponding hosts.
3) High biosecurity:
Phages are composed of nucleic acids and proteins, which can be degraded into amino acids and nucleic acids.
Unlike antibiotics, phages are important natural symbionts of humans and animals.
4) A high-level ability of proliferation:
A high-level ability of proliferation is conferred to phages, which can produce around 100 progeny phages
after infecting only a single bacterium. While
sometimes osmotic drugs are impenetrable, phage therapy can easily achieve the desired effect.
5) Wide distribution:
Phages nearly exist in every corner of ecosystems.
6) Easy to filtrate and obtain:
In most cases, desired phages can be readily obtained by artificial culture of bacteria, filtration and
purification. The time-consumption of filtrating desired
phages of its corresponding drug-resistant strains is shorter than other methods, sometimes only a few weeks
or months.[3]
7) High efficiency:
Phages are effective against multi-drug-resistant bacteria. Thus, they can be used in combination with
antibiotics for frequent synergistic effects.
8) Targeting:
Phages can be applied with high accumulation at the site of bacterial infection (a self-replication drug).
[2]
9) The genomes of some phage strains have been completely described and explained.
The genome modification, verification of gene functions, products expression and application of these phages
have been widely studied. These studies can be the theoretical bases of phage modification or rebooting, and
provide the protocols for synthetic biological operation.
However, phage therapy has not been widely used due to limitations as follow:
1) Host bacteria may be resistant to phages.
The resistance mechanisms of bacteria to phages can be divided into four categories as follow:
① Adsorption inhibition:
Bacteria can change their receptors by mutation of their own genes, such as receptor deletion, concealment
of receptors and receptor mutations, to
prevent phage adsorption; or to avoid phages approaching by producing capsule and mucus layer.
② Abortive infection:
The replication of the phage was blocked when the phage injected its DNA into the host bacteria, and there
was no release of the progeny phage.
③ Penetration barrier:
Bacteria can use anchoring protein or membrane protein on the outer membrane of itself to prevent the phage
DNA injection. Genes encoding these proteins can often
be found in prophages. The bacteria can acquire resistance to the corresponding phage when the genome of
these prophages is integrated into bacteria.
④ Degradation or interference to phage DNA:
The restriction modification system of bacteria can protect the DNA of itself. And it can cut and degrade
the external DNA after recognizing it. The
regular cluster of short palindrome repeats (CRISPR) in bacteria can also resist phages. Besides, the phage
transduction and the binding of sexual mycelium
may cause gene transfer between bacteria, which leads to the production of a new microorganism or even more
resistant bacteria.
2) Narrow host spectrum:
The host spectrum of bacteriophages is generally narrow, so it is necessary to select the corresponding
phages strictly in order to have bactericidal effect;
3) Strong immunogenicity:
As a heterologous particles, phages have strong immunogenicity, which may stimulate the immune response and
suppress the ability of bactericidal effects.
4) The effective dose and time of phage therapy are limited:
The phage could be cleared from the body by immune after 72 hours, and only when the number of bacteria
reached a certain level, could the phage proliferate.
The early inoculation or improper dose may lead to phage being removed before it can play a role;
5) The toxin gene:
The toxin gene carried by phages may cause some adverse reactions, so we should avoid using phage with
transduction effect;[4]
6) Lysogeny transformation:
In some specific conditions, phages' genome can be changed, and not express the lysosomal substance after
entering the host cell. On the contrary, it is integrated into the host bacteria genome, and become a
lysogenic phage, which is not suitable for clinical treatment. However, to make things worse, it may make
the host bacteria obtain virulence factors;
7) The pharmacokinetic characteristics are complex.
Due to the unique proliferation and immunogenicity of phages, the metabolism of phages in vivo is not only
related to the types of phages, but also affected by the number and metabolism of bacteria and the immune
system of human body. Different phages have different pharmacokinetic characteristics. In addition, the data
we collected of phages' growth in vitro can not be directly applied to the practice in vivo, which makes it
difficult to grasp the appropriate timing and dose of administration;
8) The virulence factors carried by phages are risky.
The phage genome contains fragments that change the pathogenicity of bacteria, which may cause a series of
phenotypic changes in the host bacteria of lysogenic transformation, resulting in bacterial toxicity or
enhanced toxicity;
9) Bacterial lysis will release endotoxin.
In the process of phage therapy, not only the host bacteria in vivo will release a large amount of endotoxin
after being lysed by phages, but also it is difficult to avoid the mixing of bacterial endotoxin in the
cocktails and purification of phage preparation, which makes the use of bacteriophage system in vivo harder.
10) High cost.
The high cost of the collection, maintenance of large libraries of different bacteriophages makes safety
testing more difficult and expensive.
By using gene engineering and synthetic biology technology, artificial modification, construction and
rebooting of non-natural phages can solve these problems. For problems such as lack of natural phages
resources, the difficulties in breeding of valuable characters, the narrow host spectrum of phages, easy to
be eliminated by human immune system, and evolution of bacterial resistance to phages, modification at the
gene level can effectively ameliorate this situation.
For example, in 2019, Helen Spencer's team improved the bactericidal capacity of a Mycobacteria phage strain
by genetic modification. They applied this phage to experimental treatment, and achieved clinical success by
this treatment method. In 2007, Timothy K. Lu's team added gene DspB to T7 phage, which can degrade biofilms
and improve antibacterial ability. In 2015, Hiroki Ando's team genetically modified T7 phage to enable it to
recognize new host cells. Although there are many studies on phage gene modification, it still lacks a
general efficient and accurate method to modify phage genes. This situation has hindered such researches.
Therefore, we proposed our project to solve this problem.
Our initial idea is to produce phage by synthetic biology. The basic idea includes fragment the phage
genome, and modify its genes, then reassemble and reboot it. To make the fragmented phage gene work, we
thought about rebooting the phage. Inspired by the idea of artificial yeast chromosome (YAC), our we planned
to amplify the large-scale genome of a virulent phage by PCR according to its functional gene cluster, and
then assemble the amplified small fragments with the chromosome skeleton of artificial yeast to form
multiple plasmids The genomic library was constructed in yeast. After modifying the genomic library of the
virulent phage to our expectations, the plasmid carriers of the whole genome were introduced into the host
bacteria, and then they were expressed and assembled separately to produce new phages. This platform makes
it possible to simultaneously edit or simplify multiple gene loci encoding specific functions of large-scale
phages, so as to produce active engineered our "perfect" phages.
References
[1]Zou Xiuyue & Cai Dezhou. Research progress and development direction of bacteriophage therapy for
bacterial diseases. Chinese Journal of infection control (2019)
[2]Gu Yuanyuan, Hua Guodong, Gong Ying, GUI Yue & Han Shaxi. Research progress on in vitro antibacterial
activity of Chinese herbal medicine and its active ingredients. Chinese pharmacoeconomics 13, 123-125 (2018)
[3]Zhu Yuwei et al. Research progress of bacteriophage and its treatment of bacterial infection. Chinese
animal husbandry and veterinary 42, 769-773 (2015)
[4]Nikolich, M. P. & Filippov, A. A. Bacteriophage Therapy: Developments and Directions. Antibiotics 9
(2020).
Phage researcher
Interview reasons:
The researchers have a professional understanding of the characteristics and advantages of phage itself, as
well as the limiting factors for the application and promotion of
bacteriophage therapy. We hope to ensure that our ideas are meaningful and valuable by interviewing and
communicating with them, as well as guide our project to solve the problems in bacteriophage therapy.
Interview contents:
Dr. Xu Yongping, professor and doctoral supervisor of School of bioengineering, Dalian University of
Technology, the director of Engineering Research Center of animal food safety
guarantee technology of Ministry of education; has certain research on bacteriophage technology in
controlling animal bacterial diseases.
Professor Xu Yongping thinks that the function of bacteriophage is almost the same as that of antibiotics,
which has four advantages. From the perspective of application history,
phage therapy has been used for more than 80 years. So far, no safety problems have occurred. From the
distribution point of view, phages widely exist in our lives, such as water, air and
hands. From the nature, bacteriophages only lyse bacteria. At present, the U.S. FDA (Food and Drug
Administration) has approved the use of some phage drugs, most of which are used
to treat intestinal diseases. In addition, phage has the advantages of low cost and fast screening.
Currently, the phages we use are all natural, and the transformed ones are in the
research and development stage. Our project may be able to promote the research of phage modification, which
is of a great significance.
Dr. Zhu Tongyu, professor and doctoral supervisor, is currently the director of Shanghai public health
clinical
center, the director of Shanghai Institute of bacteriophage and drug
resistance, and vice president of Zhongshan Hospital Affiliated to Fudan University. In recent years, he has
paid special attention to the prevention and control of infection after
transplantation. Since 2018, he has presided over the first clinical trial of phage therapy for super
resistant bacterial infections in China.
Professor Zhu Tongyu is carrying out a clinical trial of bacteriophage therapy in the prevention and
treatment of drug resistance instead of antibiotics. He told us that there have been successful cases of
treatment. Compared with antibiotics, he believes that the greatest benefit of bacteriophages is to minimize
the demand for antibiotics, while humans can find a balance with nature. Phage therapy has little side
effects on human body, and its action speed is fast. However, the main problems are that the phage therapy
may cause host tolerance , the phage library that can be used for medicine currently is not large enough,
and the processing, culture and amplification of medicinal phage is relatively time consuming, which limit
the popularization and application of phage therapy. He said that the use of synthetic biology can improve
in many aspects, such as considering the combination of lysogenic and lytic cycle related genes to realize
the regulation of lytic rate.
Dr. Tong Yigang, Professor of molecular microbiology, director of Department of omics and bioinformatics,
State
Key Laboratory of pathogenic microorganism biosafety, Institute of microbial epidemiology,
Academy of Military Medical Sciences. He has taken the lead in undertaking a number of national 863
projects, including microbiology, super resistant bacteria, bacteriophage, etc.
Professor Tong Yigang told us that bacteriophage, as a relatively new research direction, has a very
important reason that its basic research is relatively small. If we can understand the mechanism of
replication, infection, packaging and control genes of phages, it will be very useful for the design, use
and modification of phages. But phage still has many problems while its application range is
narrow. Although it is safe and does not damage the flora, it needs targeted treatment, which leads to high
cost and is not suitable for promotion. He told us that we could use our project to design,
synthesize and transform phages from multiple sites. Combining the advantages of the project and the
forefront of bacteriophage research, we should make some attempts, such as enhancing the broad spectrum,
tolerance, bactericidal ability and survival ability of phages before being transported to specific sites
for use, which will play an important role. In the process of our project implementation,
we combined the teacher's suggestion to our project design.
Dr. Li told us that antibiotics are easy to destroy the normal flora of human body in the process of use,
but phages are highly specific, which does not harm the normal flora of human body. Nowadays the main
problem of bacteriophage is that it is easy to produce resistance in the process of use. There are some
methods to solve this problem, such as site directed mutagenesis of genetic engineering, lyase, cocktail and
so on. Reconstruct phage may solve these problems such as narrow host spectrum and host resistance, but the
engineered phage may cause security concerns or safety hazards. The current use of natural phages has
resource limitations, and the producing of engineer phages has many challenges. She suggested that we
should carefully consider the safety of synthetic phage in the project design.
Mr. Li told us that antibiotics are easy to destroy the normal flora of human body in the process of use,
but phages are highly specific, which does not harm the normal flora of human body.
Nowadays the main problem of bacteriophage is that it is easy to produce resistance in the process of use.
There are some methods to solve this problem, such as site directed mutagenesis of
genetic engineering, lyase, cocktail and so on. Synthetic phage can solve the problems of narrow host
spectrum and resistance of phage, but the restricted synthesis and transformation of phage
is still its security problem. The current use of natural phage has certain limitations, and the synthesis
is more difficult. She suggested that we should carefully consider the safety
of synthetic phage in the project design.
What did we get?
Through interviews with bacteriophage researchers,
we know that bacteriophage has the advantages of wide distribution, strong specificity,
no damage to normal flora, and rapid action. It is ideal to be used as an ideal alternative to antibiotics.
Most of the phages that can be used are natural phages now. However their narrow host spectrum and easy
tolerance restrict their application. We think it is of great significance to reconstruct phages and improve
their shortcomings to get the "perfect" phages.
Bacteriophage medicine developers
Interview reasons:
Bacteriophage medicine developers focus on the research and development of bacteriophage drugs. They have a
clear understanding of the research
and development and demand of phage products. In addition, they are more aware of the problems in the
process of research and development of phage products.
We hope that through interviews, we can understand more current research methods of bacteriophage products
and the problems that are urgent to solve
in the process of research and development, so as to think about the potential of our project Enough to
solve their problems.
Interview contents:
Noanbert Biotechnology Co., Ltd. is a bacteriophage drug R & D provider, invested and established by
entrepreneurs and professors who have served
China's animal health industry for many years. It focuses on the development of bacteriophage veterinary
drug products in the field of animal protection,
and is committed to developing bacteriophage products as an alternative drug for antibiotics, so as to
provide solutions to bacterial diseases for the healthy
development of China's planting and breeding industry.
Dr. Wang, a senior scientist from the Technology Department of Qingdao Nova Biotech Co., Ltd told us that
they had developed a product named noamcinolone for the diseases caused by E.coli and
Salmonella in poultry.
The mixture therapy with bacteriophage cocktail has achieved good results in clinical practice and is highly
praised by the farms. However, because of the activity of phage, its survival and production conditions make
the use of phages is more complex than that of antibiotics. The metabolic time in vivo may be shorter than
that of antibiotics. It may be cleared by the immune system.And there is also the problem of narrow lysis
spectrum. He expected that our project could modify phages to solve these above problems and he also suggest
us to design and produce heat tolerant bacteriophages.
Qingdao Runda Biotechnology Co., Ltd. is a key enterprise integrating research and development, production,
sales, technical services and information
services of veterinary drugs and feed additives. All kinds of veterinary drugs and feed additives developed
by the company take the effect as the premise
and reduce the breeding cost as the principle, which has been widely recognized in the market.
Mr. Zhai, the director of the technical service department told us that bacteriophages are non-antibiotic
products. Phage therapy can reduce bacterial resistance and antibiotic use, which is more in line with the
current national policy. However, it has the problem that the host specificity is narrow. It is promising to
construct a phage library available for screening or to produce phages according to customers need. Besides,
the Chinese government does not approve any bacteriophages as feed additives or veterinary drugs. He also
suggests us to remove the uncertain and harmful factors of phage gene through gene editing in the future,
which will be more targeted and safer and promote the development of phage product industry.
What did we get?
There are few manufacturers of phage products in China now. In the process of studying phage products, these
manufacturers need to
consider the survival conditions of phage products in many aspects because of their biological activity.
Also, phages have strong specificity,
and the screening process is complex. In addition, due to the lack of relevant national legislation on phage
products, there are no clear provisions thus
the related experiments can not be carried out. This also makes us think further that we should not only
improve the technology itself, but also consider
the national legislation, policy and other aspects. In our later practice, we also incorporated the content
of legislation into our activities to promote national
legislation and provide institutional guarantee for phage therapy.
Conclusion
Through interviews with bacteriophage researchers, bacteriophage manufacturers and others, we know that
phages are wildly concerned for its wide distribution and rapid function without destroying human intestinal
flora. In addition, relevant experiments also have been carried out successfully in human clinical, animal
breeding and other aspects. But due to the narrow host spectrum which may easily cause resistance, as well
as the national policy, the phage therapy has not been widely accepted. We believe our project is of a great
significance. It can provide solutions for the transformation and production of bacteriophages that are more
in line with the medical and safety requirements. Besides, we also start to think about how to promote
national legislation to further promote the research and development of bacteriophages at the same time.
6 The Third Stage: Feasibility Analysis
Expert interview
Interview reasons:
Experts in the field of phage research and synthetic biology have a deep understanding on phage and
synthetic biology methodology. So, we hope that our project will become feasible and reasonable with their
guidance.
Interview contents:
Dr. Lili Wang, associate professor and master supervisor of School of bioengineering Dalian University of
Technology. Her major research interest is control of animal bacterial diseases by lytic phages.
She suggested choosing proper insertion site would be particularly important. In addition, whether the
insertion will affect the one-step growth curve and lysis effect of the phage needs to be further
determined. She thought the idea and design were very good, but it would
be difficult without foundational experiments in the early stages. There would be a lot of uncertainties in
the process of the experiment. And it may take a long time to select the sites, so she suggested us to start
from the transformation of a single phage and get familiar with the knockout system to equip ourselves with
certain foundation and experience.
Shengjian Yuan
Ph.D Candidate
M.S. Bioengineering, Sun Yat-Sen University
Mr. Yuan Shengjian, Ph.D Candidate of Sun Yat-Sen University. He obtained his master degree in
bioengineering. His research interests include artificial phage synthesis, gene function identification,
modular design and synthesis of phages.
During the whole season, we carried out our communication with Dr. Shengjian Yuan about our project. Dr.
Yuan believed that it is necessary to reconstruct phage genome and reboot phage particles, especially the
lytic phages with giant genomes, because it is obvious that large genome contains longer gene circuit.
However, he also told us that de novo synthesis of large gene fragments and manipulate the genome is
still
technically difficult. For example, it is technical difficulty to introduce T4 genome (168kb) to its host,
E. coli. Since the plasmid fragments plan to be used were too large, the efficiency of ordinary
electroporation might be relatively low.
Inspired by Dr. Yuan, we began to model the electroporation of competent E. coli with large-sized
plasmids
and started thinking about how to get enough T4 whole genome fragments for manipulation. Dr. Yuan told us
common and alternative methods for synthesizing and manipulating phage genomes mainly include yeast
recombination platform, which can manipulate huge genome fragments of Mbp level, but the difficulty lies in
the extraction of large plasmids after genome construction and host transformation.
He told us that the current PCR technology can amplify fragments within 20 kbp at a time. The CRISPR/Cas9
system is suitable for gene knockout and insertion. Considering the cost issue, we finally chose to amplify
the genome of T4 phage by PCR, and then splicing and circularized the genome fragments with linearized YAC
vectors by the method of gap-repair cloning in yeast.
Dr. Yuan also suggested that in the future, we can consider redesigning the phage genome, such as
modularizing the genome arrangement and trying to construct the smallest genome.
Dr. Wu Yi: Associate Professor and doctoral supervisor, School of Chemical Engineering, Tianjin University.
Main research direction: synthetic biology (synthetic genomics), yeast genome design and synthesis,
mammalian artificial chromosome design and synthesis, etc.
Dr. Wu first told us that the assembly of small molecule DNA mostly adopts in vitro assembly strategy, but
because large pieces of DNA are easy to break during in vitro manipulation, the assembly of large molecule
DNA is completed in vivo with the help of the host's own recombination mechanism. Since our project involved
the manipulation of a T4 phage with a 168.9kbp giant genome, we finally chose to splice the T4 genome in
vivo.
Dr. Wu told us that commonly used assembly hosts mainly include E. coli, S.cerevisiae and Pseudomonas
aeruginosa. However, our project aimed at assembling and manipulating the genome of virulent phage T4,
and
its gene products have potential toxicity to E. coli. Bacillus subcultivis as the host can
only produce
large exogenous DNA fragments with stable GC content below 44% (length greater than 200 kbp), which will
limit our proposed wide application on the platform. In the end, inspired by Dr. Wu Yi, we decided to use
S.
cerevisiae as the assembly host for large fragments of the T4 genome.
Dr. Yingfei Ma: Researcher and doctoral supervisor. His research interests include the identification of
phage genome, phage function in various ecological niches by means of bioinformatics, artificially phage
synthesis and the identification of necessary gene functions, modular design and synthetic biology of
phages.
During the interview with Dr. Ma, he convinced our current design plan. However, Dr. Ma reminded us that
although there were mature methods and strategies for the synthesis of large fragments of DNA, it was still
very difficult in practice. In other words, the experiment may have operational difficulties, but the
experimental process had no theoretical problems. After receiving Dr. Ma's advice, we decided to retain the
required bacterial colonies after each step of transformation and screening, so as to timely backtrack if
any step had problems.
Conclusion
Through our interview to the experts of phage and synthetic biology, we gradually finished the design of our
project. We halted our first design and redesigned project after interview with them. We decide to develop a
yeast-based platform to genetically manipulate and reboot Coliphage T4. We optimized our experimental design
proposal step by step to make it a feasible, easy-to-operate and safe project.
7 The Fourth Stage: How can we maximize our influence on society?
Expert Level
Future Application Direction
We are currently trying to rebuilt and reboot bacteriophages. We expect to apply synthetic biology to solve
the challenges faced in the application of natural phages, such as host spectrum is narrow, bacteria prone
to resistance, easy cause of endotoxin blood disease, etc., to enhance the broad spectrum of phage,
tolerance, sterilization ability, survival ability and other ability, and promote the research and
application of phage therapy. From lab to industry, what are the challenges for future application and how
can we maximize our influence on society?
Project Sustainability
Professor Fan Yu from the School of Foreign Languages of Dalian University of Technology has nearly ten
years of experience in
leading the Model United Nations and has a deep understanding of various United Nations documents and
backgrounds.
We had a conversation with Prof. Fan Yu. We elaborated on the purpose and reality of the project. Prof. Fan
thought that our project was in line with the basic core of SDG: people-oriented. Our project is based on
human beings, solving human problems with engineering thinking and methods, providing a convenient way for
engineering phages, which is of positive significance and value for preventing the spread of antibiotic
resistance. Our project is contributing to SDG goals in terms of health and well-being and responsible
production and consumption. But the teacher also mentioned that the biosafety issues involved cannot be
ignored, especially the potential of bacteriophages to spread resistance genes, pollute the environment and
harm the human body. The biosafety protection of SDG and the relevant documents of the United Nations on
biosafety have paid attention to related issues. We should have a deeper understanding of the connection
between SDG and the iGEM community and the connection between human beings as a species, and clarify the
responsibility and feelings of the concept of sustainable development.
Non-expert Level
Public Acceptance
We hope that our project can promote the research of synthetic phage, and act as an alternative to
antibiotics, to help solve the problem of bacterial
infections in humans and animals. In our interview with Zhu Tongyu, we realized that a major challenge of
our project was to make people let go of their
fear of the unknown and accept this new treatment. In interviews with Director Lu and Director Song of
Dalian Zhongshan Hospital, we found that the
introduction of a new treatment method often takes a long period of time, and due to concerns about safety,
moral and ethical issues, the promotion
and use of new treatment methods in the clinic was slow. The public took a conservative and hesitant
attitude towards the emergence of new things.
In order to further understand the public’s acceptance and expectation of phage therapy, how difficult it is
for phage therapy to be applied and
promoted clinically, and what factors affect the public’s acceptance of new things, we conducted a detailed
questionnaire survey. Detailed
questionnaire results and analysis can be found below.
The results of the second questionnaire
In order to further understand the public's acceptance and expectation of phage therapy, how difficult it is
for phage therapy to be applied and promoted clinically, and what factors affect the public's acceptance of
new things, we conducted a detailed questionnaire survey. In order to better reflect the opinions of
different people, we pay a attention to the diversity of people in the process of distributing the answers,
and distribute them through network, community and WeChat, etc. After dozens of days of waiting, we finally
recovered a total of 543 questionnaires, among which there were no blank answers and no invalid
questionnaires.
1. Educational background
According to the results of the questionnaire, the age distribution of the respondents is roughly balanced.
There are about 119 people with educational background below junior high school, 154 with high school
education, 140 with university degree, and 130 with postgraduate education. Although the number of the four
groups is slightly different, it is roughly in line with our expectations.
2. Age distribution
We can seen from the age distribution of the number collected that the age distribution of our surveyors is
basically uniform, and the results obtained can basically reflect the views of people of all ages.
3. Do you know about phages? [single choice]
A. Unware
B. Yes and describe it briefly________
From the overall data, more than half of the respondents did not know what the bacteriophage was while about
45% of the respondents thought they knew what a bacteriophage was.
In order to further grasp their understanding of phages, we asked those respondents who thought they knew
what a bacteriophage was to give a description of the phage in the questionnaire. We used the word cloud to
analyze the key words.
It can be seen that less than 45% of the people who answered "yes" understood that bacteriophages are the
essence of viruses, but quite a number of people mistakenly believe that bacteriophages are bacteria. In
addition, some people mentioned some keywords related to phage, such as RNA, shell, replication, etc., thus
we think that there are quite a few people who really know about phages.
4. The following are the scale questions ("Not serious at all", "Not very serious", "General", "A little
serious" and "Very serious", which are respectively divided into 1, 2, 3, 4, 5) [matrix scale
questions]
It can be seen from the table that the most people think that the abuse of antibiotics is not very serious
and A little serious, while less than 4% of the people think that the abuse of antibiotics is Not serious
and Not very serious. As we can see, people have a certain understanding of the harm of the abuse of
antibiotics. This will contribute to the promotion of phage therapy in the future.
Now let me tell you that bacteriophage is a kind of virus that can invade bacteria and must be
parasitized in living bacteria. Therefore, it can be found in some places where bacteria grow.
Bacteriophage therapy may be an alternative to antibiotic therapy to help people tide over the
difficulties. What did you think when you heard about this? [single choice]
As the table shows that more than half of the people are optimistic about phages after hearing the objective
introduction given by us. In addition, 15.5% of the people are worried about whether the phage will cause
other negative effects on the human body, and 21.71% of the people think that the nature of bacteriophages
is still a virus, and they have reservations.
6. Do you know what fields phages are mainly used in at present in China? [singe choice]
We can see from the results that although more than half of the people think they know bacteriophages, most
of them do not know the application fields of bacteriophages. Only 13.95% people think they know the
application field of phage. After the word cloud analysis of the key words, we found that the words with
high frequency were "treatment" and "drug", which showed that some people in 13.95% of the population had
cognition of phage. Besides these words, there were more vague words such as "genetic", "biological" and
"research", which were not ideal answers.
7. Can the following events make you more receptive to phage therapy? (" Unable", "Less able", "General",
"More able" and "Very able" are assigned to 1, 2, 3, 4, 5 respectively) [matrix scale questions]
By analyzing the overall response, we calculate the total score of the five expected events. We can see that
in people's minds, the government's relevant laws on phage therapy are the most promising events that can
make them have a higher acceptance of phage therapy. The second is primary and secondary education, news
broadcast, and phage manufacturers' advertising is the most unreliable expected event.
To sum up, people have a certain degree of inaccurate understanding of phage therapy, and there are some
resistance psychology from various reasons for phage therapy. It can be inferred from the results of the
recovery of the last topic that legislation may be a nice choice to improve the society's view on phage
therapy.
Industrialization of Bacteriophage Products
In our previous interviews and research, we interviewed several scholars. They all stated that in China, the
legislation on
phage is far behind the research. Teacher Tong Yigang said that the country had never approved the use of
genetically engineered
potent or lysogenic bacteriophages for clinical applications, which remained at the level of laboratory and
animal experiments,
and clinical trials were rarely carried out. Teacher Li Xiaoyu said that at present, phages were mainly used
in laboratories.
For industrialization, they still needed to be verified by experiments. However, the domestic application
for approval procedures
in China was tedious, and only a few were doing it, so we may encounter many difficulties in this regard.
Moreover, some of
the current phage manufacturers have been slow due to the lack of corresponding legislation in the country.
This aroused our thoughts. At present, the slow development of bacteriophages in China, the lack of
bacteriophage manufacturers,
and the slow progress of research has a lot to do with legislation. Although there are clear laws and
regulations restricting the use
of antibiotics in China, there are no clear regulations on phage-related products, and companies and
manufacturers expect the country
to issue relevant laws and regulations.
Legislative Proposal
Based on the questionnaire survey results, phage clinical research and the requirements of manufacturers, we
finally wrote a legislative
proposal, hoping to get the attention of national legislators and promote the research of phage therapy in
China.
For more information, please click this link.
8 References
[1]Global antimicrobial resistance and use surveillance system (GLASS) report Early implementation 2020
[2]Bulletin of the World Health Organization 2016;94:638-639.
[3]Report of Secretary General
[4]Xie Pinglin. The research progress of bacteriophages and their lytic enzymes in the prevention and
control of bacterial diseases in livestock. Foreign Animal Husbandry (Pig and Poultry),2020,40(08):59-63.
[5]Tang Ruike. Diagnosis and prevention of dairy cow mastitis. Chinese Journal of
Veterinary Medicine, 2020(6): 29-31.
Education➤
