World Studies Extended Essay

On Health and Development

BACTERIOPHAGE THERAPY:

A Global and local analysis of antibiotic resistance

integrated with the reappraisal of bacteriophages
as prophylactic alternative in Colombia

3807 words
 ACKNOWLEDGEMENTS
I give thanks to all the academic and emotional support given by my extended

essay assessor, Leidy Rocha and Paola Carvajal, both which were fundamental to

the creation of this document. I also pay special thanks to my secondary school

director, my IB director and my girlfriend which have given the faith I needed to

overcome the obstacles that had come in the process of redaction.

INDEX:
PREAMBLE.
Introduction
Methodology

I. THEORETICAL FRAMEWORK.
Antibiotic Resistance Crisis Worldwide.
Bacteriophage Physiology and bacteria mitigation.

II. ANTIBIOTIC RESISTANCE IN ALDANA

III. BACTERIOPHAGES AND THEIR EXPERIMENTAL EFFECTIVENESS

IV. THE CHALLENGES OF PHAGE THERAPY

V. CONCLUSIONS

VI. BIBLIOGRAPHY
 PREAMBLE

Introduction

The proliferation of antibiotic resistance is an increasingly relevant and delicate topic. The

strains of untreatable bacteria have raised in numbers, affecting both developed and

developing countries. The resistant pathogens have permeated society, unleashing

outbreaks that have afflicted thousands of families such as mine. Families that, under the

advice given by medics, have no other choice than to pray, as no definitive cure exists for

those who have been infected with superbugs1 (Semana, 2008). Under the pressure of that

premise, I searched for information about alternative treatments to resistant bacteria and

found about “bacteriophages”, viruses that specifically attack bacteria.

Accordingly, the research had brought me to a point where I had found a global problem

(The antibiotic resistance crisis) which lead into a local problem that developed in a

geographical location (the multiresistant strains of H. pylori, E. Coli, P. Aeruginosa in

Colombian territory); and also a local solution that used biological findings (bacteriophages)

applied on an outside territory (Bacteriophage Therapy as a treatment in Easter Europe).

As I was making the connection between these topics; I found little to none information of

their relation in my local context and thus, decided to establish the next research question

to evaluate if the situation in Aldana could be solved once and for all:

1
Bacterial strains resistant to all known antibiotic families.
 To what extent is bacteriophage therapy an effective prophylactic alternative to

antibiotics in the epidemiological context of antibiotic resistance in the global and

Colombian panorama?

Along this order of ideas, the research question implied that phage therapy may resolve the

antibiotic resistance crisis in Colombia and the World. For this disambiguation of this

assumption to take place, we first need to comprehend the development of the antibiotic

crisis and bacteriophage physiology as found in the Theoretical Framework, consequently

reviewing the epidemiologic profile of three of the most common bacteria found in the

country as seen in Chapter 1, thereupon arriving at how bacteriophages have been proven

effective around the world just as evidenced in Chapter 2, followed by the evaluation of

phage therapy inside human populations in Chapter 3. And finally closing on my

understandings of this whole phenomena and research demonstrated in the Conclusions

section.

Methodology

The current work is a systematic review on epidemiological literature destined on compiling

the demographics behind the most common antibiotic resistant bacterial strains in Colombia,

evaluating the use and effectivity of bacteriophages in vivo and in vitro conditions, and

assimilating the results bacteriophages have given on communities around the world into

the Colombian sanitary context. For this study, publications solely reported by Medline and

PubMed between the years 1970 and 2017 were checked for. The keywords used for

identifying such publications were a combination of epidemiology, Colombia, H. pylori, E.

coli, P. aeuroginosa, MRSA, bacteriophages, treatment, in vivo, in vitro, population.

Publications investigating the current Health situation of Colombia made and published by

the National Health Ministry, the World Economic Forum and the World Health Organization

were also checked for.

THEORETICAL FRAMEWORK

Antibiotic Resistance Crisis Worldwide

Infectious diseases are classified among the leading causes of death in the world and are

therefore considered a public health problem. Among them, bacterial infections are of great

concern. As their populations increasingly acquire resistance to antibiotics they are

beginning to be once again untreatable by orthodox measures, hence, posing a real threat

that is predicted to kill more than 10 million people worldwide by 2050.

Figure 1.1: Prospect of antibiotic resistant bacteria incidence in 2050

(World Economic Forum, 2014)

Dissemination of bacterial resistance could lead to the generation of problems with

enormous dimensions such as the exponential increase in mortality, morbidity, and costs of

medical care. Seconding the information given by the World Health Organization (WHO),

only in the United States are presented 99,000 deaths per year from infections caused by

bacteria resistant to antibiotics, hospitals and the cost of medical care associated with. They

oscillate between 21 and 34 trillion US dollars annually (WHO, 2017). Furthermore, the

problem is demonstrated to be delocalized; in Russia, 86.3% of households use antibiotics

indiscriminately; In Tanzania, Sub-Saharan Africa, the number of deaths per antibiotic-

resistant bacteria doubles the number of malaria deaths (World Economic Forum, 2014).

Moreover, in the case of Colombia there has been an increase in the isolation of

multiresistant2 strains, both in Pediatric Intensive Care Units (ICUs) and in adults ICU

(Salvatierra & Benguigui, 2014). Among the most dramatic increases is the case of beta-

lactamase3 producing Escherichia Coli, which because of its protein inhibition system is

made invulnerable to common antibiotics. This species saw an increase of infected in the

3% between 2012 and 2013 (WHO, 2017).

It has been postulated that the increase in resistant bacterial infection is due to the

inappropriate use of antibiotics (Department of Food Safety, Zoonoses and FoodBorne

Diseases, 2006). Common situations such as uncomplete prescribed doses or prescriptions

and unnecessary medical practices have made so that there are bacterial infections that can

be no longer cured by an antibiotic. This panorama has led governments and health

agencies recently to invest much more in consciousness campaigns about safe use of

antibiotics and the importance of developing new ones (Prada, 2008).

2
Resistant to two major antibiotics and treatable only by experimental ones.
3
Enzyme that destroys the beta-lactamase rings found in penicillin derived antibiotics, thus, making them
unviable for bacteria presenting this mutation.
 Figure 1.2: Transmission Pathways of Antibiotic Resistance

(WHO, 2017)

The source of antibiotic resistance extends well beyond the inappropriate use in humans,

there is also an indiscriminate management of these substances by the food industries. As

illustrated in Figure 1.2 in the industrial animal production sector for human consumption,

the permitted antibiotics are scarce and sometimes there is no effective treatment on the

control and prevention of bacterial pathogens (Philips, 2004). However, there are studies

indicating that more than 80% antibiotics sold in the United States and at least 50% of those

produced in China are directed as growth promoters to animals suited for human
consumption such as pigs, chickens and cows. The inclusion of such promoters reduces the

quantity of food that is supplied and induces an increase in the weight of the animal;

consequently, raising economic returns for producers (Levin Institute, 2014).

Phage Therapy, bacteriophage Physiology and bacteria mitigation.

Bacteriophages recognize the surface of the bacterial cell with high specificity, inject

their DNA or RNA, and multiply and assemble within the bacteria, to finally break the

cell and release their progeny, which will infect new bacterial cells.

The number of phages grows exponentially, and as so, their therapeutic effect

increases with time. Furthermore, the selection of new phages is a process which

occurs relatively fast (Sulakvelidze, Alavidze, & Morris, 2001). Phage therapy was

proposed from since their discovery and has been developed in countries such as

the Republic of Georgia, Poland, Russia, and the former East Germany; However,

after the discovery of penicillin and other antibiotics, research in Phagotherapy in

Western Europe and America was abandoned.

In recent years, with the increase in the frequency of appearance of multiresistant

and pan resistant strains and to antibiotics, researchers have retaken the interest in

the development of phage therapy and its implementation for the control of

contamination and bacterial infections in various fields (Kutter & Sulakvelidze, 2005).

The cycle of viral infection begins with adsorption from phage to susceptible bacteria.

The adsorption occurs in two stages, the first reversible followed of the irreversible.
The reversible interaction is weak and alterable by any change in the medium; the

irreversible interaction is, on the contrary, mediated by structures or specific

receptors. This specificity shows that a phage can only infect bacteria that have the

receptors to which the phage can be unite (Kutter & Sulakvelidze, 2005). The nature

of the receivers varies according to the phage: proteins and lipopolysaccharides of

the cell wall, teichoic acids, flagella and pili can serve as receivers. In the virus order

of Caudovirales the irreversible union is mediated by proteins of the basal phage

plate and specific receptors of the bacterial wall. The speed and efficiency of

adsorption are important aspects that vary depending on the phago-host system that

is being analyzed, external factors and the physiological state of the host. Once the

irreversible union has been achieved, the material phage gene enters the bacterial

cell, process which involves the reverse transcription mechanism in which the

phage’s RNA sequence is converted into a DNA sequence that is later on

assimilated by the host cell (Kutter & Sulakvelidze, 2005). Next, and depending on

their nature, the phages can perform one of the following two types of life cycles:

lythic (ending with the lysis of the bacterium infected), lysogenic (the phage genome

is inserted into the genome of the bacteria and does not occur immediate lysis).
 Figure 1.3: Bacteriophage Life Cycle

(WHO, 2017)

In the lytic cycle the phage multiplies within the bacterial cell, lysing it at the end of

the cycle to release new phages; the successive release of new phages allows the

infection to taint new hosts exponentially. A virulent phage is one who performs a

lytic cycle; it sticks to the bacteria (adsorption), injects its genetic material inside the

host cell (penetration) from where it directs the expression of responsible genes of

replication of the viral genome, the synthesis of new phage particles and the

subsequent lysis of the bacterium host for the release of the progeny. Lysis is the

result of damage in the bacterial cell wall, caused by enzymes specific to the phage

called lysins, which digest the bacterial peptidoglycan (Philips, 2004) .

 II

ANTIBIOTIC RESISTANCE IN ALDANA

Just from its Discovery in 1982, H. Pylori also known as Helicobacter Pylori has been

recognized as an important factor when talking about symptoms such as chronic

gastritis, stomach cancer and peptic ulcer. H. Pylori as many other bacteria, has

turned resistant to its main antibiotic family, Amoxicillin, and specimens of this

resistant strains have been found all throughout the Aldana municipality found in the

southwest of Colombia, limiting with the Ecuador border.

Figure 2.1: Municipality of Aldana found in the Southwest of Colombia

(World Economic Forum, 2014)

Aldana is characterized by being a countryside border with high levels of

dependence on agricultural and cattle raising economies. Poverty levels are high,

and standard sanitary conditions are not reached as no real potable water reaches
the houses in the zone, most of common rule areas are shared with animals, and

crops are not properly washed before arriving to households (Goodman & Correa,

1996). Conditions like this, formally expand the transmission rates of the bacteria

which is often found in animal fecal residue that is thrown into the riverside, water

which is consequently used for the sustenance of the crops that people normally eat.

On the other hand, the constant interaction between species of sheep and humans

and the almost non-existent regulation of antibiotics in the territory condemns the

society to high mutation rates of the H. Pylori bacterium and consequent prevalence

of antibiotic resistance.

The numbers state that nearly 62% of all men between ages 18-65 and 63% of all

women in the same age condition, suffered from peptic ulcer due to H. Pylori in 1992

in this zone. Regarding children, the situation is even more worrying, as the zone

reaches a high standard compared with other territories as seen in the table below.

(Goodman & Correa, 1996)

 III

BACTERIOPHAGES AND THEIR EXPERIMENTAL EFFECTIVENESS

In Colombia, bacteriophages and phagotherapy have been investigated in projects

developed by the Environmental Microbiology and Bioprospection Group -MAB, of

the Universidad de los Andes. Initially, phage isolations were carried out against

strains of Pseudomonas aeruginosa resistant to multiple antibiotics (MDR) from the

Federico Lleras Acosta Hospital in Ibagué. P. aeruginosa is a gram-negative

proteobacterium, opportunistic pathogen, frequently isolated in hospitals, and

responsible for serious nosocomial infections. The effectiveness of the phages was

evaluated in vitro, inoculating the phage in the exponential stage of bacterial growth.

As a result, an immediate decrease was obtained in the bacterial population. P.

aeruginosa is capable of forming complex biofilms, which are even more resistant to

antimicrobials than planktonic cells; for this reason, the effect of both individual and

cocktail phages on biofilms formed by P. aeruginosa was evaluated. The results

obtained showed in all the cases (treatment with the individual phages and with the

cocktail) the removal of the biofilm; the most efficient was the phage cocktail (Kutter

& Sulakvelidze, 2005). Subsequently, the optimal phage dose was determined;

using reverse engineering and with stochastic simulations, an optimal phage dose

value was found against P. aeruginosa that efficiently decreases the concentration

of bacteria and prevents the development of resistance (Sulakvelidze, Alavidze, &

Morris, 2001). The optimal dose determined in silico was evaluated in vivo and in
vitro; In in vitro tests, the ability of a phage to control several strains of P. aeruginosa

MDR was evaluated by infection curves, and in vivo tests were to test the effect of

phage on infection induced in mice by a P. aeruginosa strain MDR

In the bacterial population phages showed to be effective in vitro and behaved as

predicted by the computational models, in vivo a survival of 85% of the infected mice

was observed, compared with 0% of survival in the inoculated control groups only

with bacteria (Donado, 2010) .The characterization of the phages against P.

aeruginosa included the estimation of the size of its genome by means of pulsed-

field electrophoresis (PFGE) and its sequencing, assembly and annotation (Donado,

2010). Other work in phagotherapy has been advanced in collaboration with the

Center for Aquaculture Research of Colombia (CENIACUA). The Pacific white

shrimp, Litopenaeus vannamei, is the main aquaculture product in Colombia and is

severely affected by infections caused by bacteria between Vibrio harveyi and

Bacillus licheniformis, which cause significant economic losses for the sector.

Because it is a product for human consumption, it is not allowed to use antibiotics to

treat infections for this reason and looking for another control strategy, alternative

control of bacterial infections work began on the isolation of specific bacteriophages

against the bacteria mentioned. Three bacteriophages were isolated against Vibrio

harveyi and the effect of them was evaluated individually and in cocktail against V.

harveyi strain CV1, and their evolutionary and co-evolutionary interactions were

analyzed.

The results obtained showed that when using the cocktail with the three phages the
appearance of bacterial resistance was controlled in a considerable way and the

population of bacteria remained sensitive to the action of the phages during the time

evaluated. In contrast, with the use of individual phages, resistant bacteria

dominated the culture in the first 48 hours of co-evolution with the phage. In the case

of B. licheniformis, a Gram-positive bacillus responsible for outbreaks of mortality in

L. vannamei, a phage was isolated that was tested in vitro, by curves of infection,

and in vivo in the shrimp via a reverse gastric tube. Both assays showed a good

capacity for reducing bacterial concentration and mortality in the animals,

respectively. Reemergent outbreaks caused by this bacillus have led to the

existence of a permanent source of contamination in the production system. One

possible explanation may be that the bacterium forms biofilms in the pipes of the

ponds; the dispersion of the biofilms can lead to the shrimp culture ponds being

constantly exposed to pathogen inoculums and this will lead to the reappearance of

the infection. In order to evaluate if the phages would control the biofilms of the

bacillus, different phages were isolated against the bacteria and one of them was

evaluated in a model system. The results showed the inhibitory effect of phage

suspension in the biofilm formation of B. licheniformis (Barbosa, 2013).

 IV

THE CHALLENGES OF PHAGE THERAPY

Phagotherapy has been studied and proven for many years; However, there are

some aspects that have hindered its application in the West. Despite the trials and

reports on the effectiveness of phages in several countries, in the West there is still

a need for more experimental tests to ensure their efficiency and safety to be applied

in humans. One of the most important challenges to bring the application of

phagotherapy to reality is the distrust of Western society; After all, they are viruses

that have always been recognized as harmful to health. To overcome this difficulty,

it will be necessary to carry out extensive coverage information campaigns based on

the results of scientific studies that show the advantages and limitations of

phagotherapy (WHO, 2017). The elimination of the risk of lysogenic conversion will

be decisive for the long-term success of phagotherapy. To avoid it, only lytic phages

should be selected, in order to control the possible transfer of genes that increase

the virulence of the bacterial strains, and to know the genome of the candidate

phages to be used in phagotherapy to guarantee the absence of genes associated

with virulence. and with lysogenic cycles; The latter requires significant efforts in the

characterization of the large number of hypothetical proteins that result from the

annotation of viral genomes.

There is also fear of the appearance of mutations in the phages that allow them to

infect other beneficial bacteria. According to a comparative study among the three

families belonging to the order Caudovirales, the phages that have a higher host

range are those belonging to the Myoviridae family, capable of infecting even

different bacterial genera, while those of the narrower range belong to the family

Podoviridae, which generally infect only one strain of its host bacterium (Donado,

2010). This information shows that it is possible for a phage to infect more than one

bacterial species. Wild phages have also been found that infect several species of

Gram-positive bacteria; although little is known about the receptors in this group of

bacteria, they are considered less diverse than those of the Gram-negative bacteria.

Gram positive phages are mostly bound to teichoic acids (Donado, 2010), and it is

known that the presence of N-acetylglucosamine or D-glucose is essential for its

adsorption. The teichoic acids are specific for each species, but different bacteria

can present similarity in the composition of these acids, which would explain the

activity of broad spectrum phages. In Gram-negative bacteria there is a greater

diversity of receptors; the question would be then, how often do the mutations

required for a phage to extend its host range occur in a natural setting? This remains

an unknown and requires experimentation in microcosms that resemble the

conditions that the phage will find, since not only changes would be needed in the

receptors but also the adaptation of all the mechanisms of cellular manipulation and

viral reproduction in the new host.

The conservation of phages is one of the main methodological challenges that

present themselves today. It has been shown that, depending on the type of phage,

as well as the host it has, some bacteriophages maintain their activity for longer

periods of time than others, preserved under the same conditions. Conservation

methods have been tested at 4ºC, -80ºC and by lyophilization. The methods of

lyophilization and refrigeration are, apparently, the best way to store and conserve

them (Kutter & Sulakvelidze, 2005). However, this is a subject that still requires

investigation. Regarding the regulations, there are no specifications on the

requirements that a pharmaceutical product based on bacteriophages must fulfill in

the Pharmacopoeia of the United Mexican States, in the United States

Pharmacopeia (USP) in the United States FDA or in the EFSA of Europe. Currently,

those who commercially produce phages, seek that their preparations comply with

the requirements applicable to biological products, such as purity and action

potential, among others . In the opinion of the authors of the present review, in

addition to the criteria used to date, it is essential to perform the genomic analysis of

the phages to rule out the presence of genes associated with pathogenicity and

lysogenic cycles. It is also important to design cocktails that make it possible to avoid

the appearance of resistant bacteria for as long as possible, thus guaranteeing the

usefulness of phage therapy. The lack of regulations hinders investments in the

development of Phagotherapy, so it becomes important.

 V

CONCLUSIONS

Phagotherapy has been proven to be an antimicrobial treatment with a great

potential for the control of infections and bacterial contamination in people, animals,

human consumption products and agricultural capital between others. Since their

discovery, bacteriophages have been used with therapeutic ends and have proved

to be effective both in vitro, in vivo, and in human populations without producing

important numbers of side effects and toxicity. Yet, despite the history of the

treatment going on for more than a decade, the lack of worldwide scientific support

and data has been decisive on the non-consolidation of phage therapy as a

prophylactic alternative, hence making the situation harder in Colombia. Registries

of phage metabolism and stability need to be compiled in different conditions that

could happen in a human body, also about the different interactions they have with

the host cell, ad about the answer mechanisms phages have against antimicrobial

resistance.

In the case of Colombia; the information and practices are scarce. Although research

on the use of native bacteriophages for methicillin-resistant strains of both

Salmonella and H. pillory in vitro exists and that even in vivo demonstrations such

as those discussed in chapter III with mice and shrimp have been postulated; The

literature on the use of this therapy in afflicted populations such as Aldana is non-

existent, or at least it has not been published. It is also clear that there are still no

models for the proper cultural introduction of treatment in skeptical countries such
as Colombia; and that any analysis made in this document refers to a

homogenization of the cultural features in common between the example of

Ukrainian and Colombian society; however, this is only a very limited approximation

to how it could really be addressed, but for this the political and scientific will of our

communities is needed.

So, I do not see close to the appropriation of therapy in the coming years, because

the political and economic will weigh more on scientific proposals, than the scientific

proposals themselves. Meanwhile, I do not think the situation in Aldana is solved

effectively; and much less that my grandmother can be cured of her infection. For

therapy is effective as an alternative to prophylaxis; it is not so much as a product in

pharmaceutical systems. Then a) Greater investment in the creation of a globally

unified bacteriophage library is required to understand which phages are viable

before which infections and which cocktails are optimal b) Research related to the

regulation of bacteriophages as a product and to the prevention of a crisis of

resistance to phages as it is considered in chapter II c) Systems for the cultural

approach of this treatment that point to the breaking of stigmas related to viruses in

developing countries like Colombia.

References

Barbosa, C. (2013). Co-evolutionary dynamics of the Bacteria Vibrio Sp. CV1 and phages V1G,
V1P1, and V1P2. Scielo.

Department of Food Safety, Zoonoses and FoodBorne Diseases. (2006). Antimicrobial Use in
Aquaculture. World Health Organization , 5-6.

Donado, G. (2010). Prevalence, resistance patterns and risk factors for antimicrobial resistance.
Scielo.

Goodman, K., & Correa, P. (1996). Helicobacter pylori Infection in the Colombian Andes: A
Population-based. American Journal of Epidemiology, 290-299.

Kutter, E., & Sulakvelidze, A. (2005). Bacteriophages: Biology and Applications. CRC Press, 527.

Levin Institute. (2014). Regulations of antibiotics in animals. Med, 35-40.

Philips, C. (2004). Does the use of antibiotics in food animals pose a risk to human health? A critical
review of published data. Journal of Antimicrobial Chemotherapy, 28-52.

Prada, G. (2008). Impacto de la resistencia de los antibióticos en el desarrollo de la medicina

contemporánea. Med, 12.

Salvatierra, R., & Benguigui, Y. (2014). Resistencia Microbiana en las Américas: Magnitud del
problema y su Contención. Nuevos Aires: Organización Panamerican de Salud.

Semana. (2008). Deadly superbugs now in Colombia. Semana.

Sulakvelidze, A., Alavidze, Z., & Morris, G. (2001). Minireview - Bacteriophage Therapy.
Antimicrobial Agents and Chemotherapy. Med, 649-659.

WHO. (2017, October 22). World Health Organization. Retrieved from

http://www.who.int/mediacentre/factsheets/fs194/es/

World Economic Forum. (2014, January 12). World Economic Forum. Retrieved from
https://www.weforum.org/agenda/2016/09/antimicrobial-resistance-is-the-next-global-
commons-issue/