Table of Contents

1. Introduction.................................................................................................................................2
1.1 Colibacillosis in Broiler Chicken Farms..............................................................................3
1.2 Antibiotic Resistance in Poultry Farming............................................................................4
1.3 Pathogens Causing Colibacillosis................................................................................................7
1.4 Pathophysiology of Avian Colibacillosis.....................................................................................9
1.5 Impact of Colibacillosis on Poultry Production in Pakistan.......................................................11
1.6 Bacteriophages as Therapeutics for Colibacillosis.....................................................................12
1.7 Cloning of endolysins of Bacteriophages...................................................................................15
1.8 Control and Prevention of Colibacillosis...................................................................................17
1.9 Alternative treatment options for Colibacillosis........................................................................18
1.10 Significance of the Study..................................................................................................19
1.11 Aims and objectives.........................................................................................................20

1
 Isolation and Characterization of Bacteriophages
against Avian Pathogenic E. coli
Chapter 1
1. Introduction
Antibiotics are absolutely the most important treatment for many infectious diseases,

but the major global public health issue is the growth of antimicrobial resistance (AMR) in

both human and veterinary medicine. AMR occurs when bacteria evolve mechanisms to

survive or resist the effects of antimicrobial drugs making infections harder to treat. This

results in increased risk of disease transmission and mortality among the organism (Ahmad et

al., 2021). It is anticipated that, if the situation is not addressed properly then it can result in

higher rates of death among humans, substantial financial losses, and a sharp decline in

livestock production by 2050 (Orubu et al., 2020). One of the main causes of AMR is the

excessive and inappropriate use of antibiotics in the production of broiler chickens. Antibiotic

use, which is commonly used for therapeutic purposes, disease prevention, or growth

promotion, can result in the selection of resistant bacteria in the gastrointestinal tract of

broiler chickens. In addition, humans can also acquire these bacteria through environmental

exposure, ingestion of contaminated meat, or direct contact (Ribeiro et al., 2023). Meat and

other food products are essential components of the human diet. The last 50 years have seen a

more than 4-fold increase in meat consumption, indicating a rising demand for this food

(Ritchie et al., 2017). Among the resistant bacteria found in broiler farms are Enterococcus

species and Escherichia coli, which are common gut flora in chickens but can also lead to

opportunistic infections. These bacteria have demonstrated resistance to many antibiotic

families, such as tetracyclines, macrolides, aminoglycosides, and fluoroquinolones (Abreu et

al., 2023).

2
1.1 Colibacillosis in Broiler Chicken Farms

Colibacillosis is common bacterial disease, caused by the avian pathogenic Escherichia coli

(APEC). Reduced productivity, high treatment expenses, and a significant financial burden

have all been linked to colibacillosis (Fancher et al., 2020). It is considered one of the top

four economic diseases impacting the global poultry industry. Colibacillosis affects chickens

of all ages but the disease occurs most frequently in 3-12 week old broiler chickens. The

morbidity and mortality rates due to colibacillosis may reach 60-80% and 5-50%,

respectively, which can result in huge economic losses to the poultry (Swelum et al., 2021)

Colibacillosis is a serious infectious disease that infects different animals including

humans, cattle, and poultry. The virulence factors present in E. coli strains are responsible for

their pathogenicity, which results in a variety of clinical symptoms. Colibacillosis in acute

form can results in septicemia resulting in sudden death. More commonly, chickens exhibit

subacute symptoms like pericarditis, perihepatitis, airsacculitis and other inflammatory

lesions. Key gross pathological lesions include fibrinous exudate covering various organs and

fluid accumulation in the peritoneal cavity. However, definitive diagnosis requires bacterial

isolation of E. coli because other opportunistic bacteria can cause similar lesions

(Ergashevich & Salimovich, 2022).

The disease is characterized by generalized septicaemia along with perihepatitis,

airsacculitis, pericarditis, salpingitis, and swollen head syndrome (Sorour et al., 2020).

Various predisposing factors are involved including overcrowding, vaccination stress, poor

ventilation, stress due to extreme temperatures, mycotoxin contamination in feed,

immunosuppressive viral diseases, and high level of environmental ammonia. Ventilation

issues and high stocking densities are major contributing factors on commercial broiler

3
chicken farms as they lead to elevated levels of dust and ammonia which damage the

respiratory tract and facilitate bacterial infection (Panth, 2019).

Effective biosecurity measures are crucial in preventing colibacillosis outbreaks.

These include cleaning and disinfection of housing between flocks, preventing exposure to

potential sources of E. coli through personnel, equipment or wild birds, maintaining optimal

housing temperatures and air quality, regular microbiological monitoring, and vaccination.

Numerous antibiotics have been used, including aminoglycosides, tetracyclines,

sulfonamides, β-lactams (penicillin), and fluoroquinolones. However, antibiotic-resistant E.

coli has emerged as a result of the careless use of antibiotics. So, antibiotic therapy may help

limit mortality but antibiotic resistance is an emerging problem. Therefore, various concerns

have been raised regarding extensive use of the antibiotics (Aberkane et al., 2023).

There has been a surge in the use of antibiotics over the last ten to fifteen years.

However, E. coli has become resistant to a variety of antibiotics. The devastating avian

disease caused by E. coli has had a severe impact on Pakistan's economy. Researchers are

making every effort to treat this deadly illness, usually with antibiotics, but the complications

have resulted from the widespread use of antibiotics. Antibiotic-resistant bacterial strains

have emerged for diseases affecting poultry (Moradigaravand et al., 2018). It was observed

that resistance initially emerged against penicillin (both β-lactam and non-β-lactam), and that

methicillin then emerged as the preferred medication.

1.2 Antibiotic Resistance in Poultry Farming

In Pakistan, the livestock and agricultural sectors get contributions from the poultry

industry of 11.5% and 6.4%, respectively. The poultry industry relies heavily on antibiotics to

treat and prevent bacterial diseases like colibacillosis. However, evidence shows extensive

4
antibiotic usage in poultry causes in the process of antibiotic-resistance in bacteria. The

humans can also become resistant to antibiotics by direct contact with chicken or indirectly

through the food chain. This resistance poses challenges for effective disease treatment and

control, necessitating alternative antimicrobial strategies (Sevilla-Navarro et al., 2022).

One major problem has been that administering small doses of antibiotics to these

animals greatly increases the probability of antimicrobial resistance. In the above context, a

larger portion of the antibiotics used are almost same or substitutes of antibiotics that are used

during the treatment of humans (Van et al., 2020). The misuse of antibiotics in developing

countries has become an unavoidable risk to antimicrobial resistance and also poses great

threat to the animal farming industry (Nayiga et al., 2020). Antibiotics are administered in

feed or water, often at subtherapeutic levels over long durations to promote growth and

prevent infections. However, low-level exposure facilitates resistance development as

susceptible bacteria die off while naturally resistant ones persist and reproduce (Serwecińska,

2020). Multiple studies demonstrate greater antibiotic use on farms correlates with higher

rates of resistant E. coli strains from poultry.

Subtherapeutic antibiotic doses are usually given to chickens in order to boost growth

and balance stressful conditions, as well as to treat infectious diseases and control

colibacillosis. Antibiotic overuse or misuse may cause resistant bacterial strains to arise and

proliferate. However, uropathogenic E. coli and E. coli that causes newborn meningitis are

two more extraintestinal pathogenic E. coli strains that cause illnesses in people that are

closely linked to the APEC strains recovered from chickens (Najafi et al., 2019). The

existence of APEC strains poses a serious risk to human health since they serve as a reservoir

of virulence and resistance genes for other E. coli strains (Thornber et al., 2020).

5
 The use of bacteriophages in poultry veterinary care and the poultry business has

attracted an extensive amount of research and interest. As a result, new companies creating

phage preparations have emerged on a global scale. Figure 1.1 illustrates phage applications

in poultry farming, processing, and production.

Figure 1.1. “Phage Applications in Poultry Farming, Processing, and Production”. In the
poultry industry, the Bacteriophages finds its applications in phage therapy, food biocontrol,
and disinfection (Żbikowska et al., 2020).

The antibiotic use and resistance patterns observed in poultry farms have also

emerged in bacterial isolates from humans. The research reported that genetically analyzed

resistant E. coli from both poultry products and infected urinary tracts, finding resistance-

encoding regions moving between the bacteria from chickens to humans (Aworh et al., 2021).

Another study reported indistinguishable quinolone-resistant E. coli isolates from poultry

products and human bloodstream infections. As such reservoirs of antibiotic resistance

establish in poultry farms and enter the human population, standard antibiotic therapies

become less effective against resistant superbugs. For example, modern fluoroquinolones are

losing efficacy against previously susceptible E. coli causing urinary tract and intestinal

infections. This underscores the need for better antimicrobial stewardship in food animal

production (Aworh et al., 2023).

6
1.3 Pathogens Causing Colibacillosis
Colibacillosis is an infectious disease caused by Escherichia coli bacteria. E. coli is a

Gram-negative bacteria and also a member of the family Enterobacteriaceae. The primary

pathogen carry many virulence plasmids, which causes high mortality rates in chicken flocks

(Kim et al., 2020). The micro flora of poultry typically constitutes a broad group of bacteria

known as E.coli, which can be found on their skin and feathers, in their upper respiratory

tracts, and throughout their digestive systems. Its symbiotic inhabitance in the digestive tracts

of animals, especially birds, and its extensive distribution through feces make it an important

species of bacteria in the human-animal-environment triad (Islam et al., 2021). This

widespread distribution and capacity to carry numerous genes in mobile genetic elements

make this specie a target for regular investigation as a measure of antimicrobial treatment

resistance. These mobile genomic elements provide other bacteria with antimicrobial drug

resistance determinants (Daneshmand et al., 2019)

Several serotypes and pathotypes of E. coli have been associated with colibacillosis in

animals and humans, most notably enteroaggregative E. coli (EAEC), enterotoxigenic E. coli

(ETEC), enteropathogenic E. coli (EPEC), and Shiga toxin-producing E. coli (STEC) (Smith

& Fratamico, 2017). APEC trigger extraintestinal and systemic infection in poultry, inducing

lesions. APEC uses diverse virulence genes that enable respiratory and systemic

dissemination. Numerous investigations revealed that Escherichia coli is 60–100% resistant

to common antibiotics like gentamicin, erythromycin, tetracycline, amoxicillin, and

chloramphenicol. Certain antibiotics, such as kenamycin, tylosin, novobiocin, polymixin,

amoxiclav, amikacin, and cephalothin, have been found to have good inhibitory actions in

other studies. However, these antibiotics have not yet been thoroughly studied.

7
 The focus of treatment shifted to using vancomycin after the development of

methicillin resistance. As expected, some strains of E. coli have since developed resistance to

vancomycin. Such resistance in an infected poultry can have disastrous financial effects

(Saeed et al., 2023). It was believed that the large antigenic diversity among strains of avian

pathogenic E. coli was influenced by the geographic location (Ibrahim et al., 2022). Recent

research has examined the role of specific virulence genes and pathogenicity islands in

causing colibacillosis. A recent study examined 150 E. coli isolates from chickens that died

of colibacillosis to check the presence of 17 virulence genes associated with diarrhea,

septicemia, or avian pathogenic strains. They found 7 genes were most prevalent in these

isolates including fimC, iucD, tsh, fyuA, irp2, papC, and astA. Over 50% of strains had some

combination of the iron acquisition systems fyuA/irp2 and disease markers iucD, tsh, fimC

pointing to their key role in pathogenesis (Sarowska et al., 2019).

It is quite important to understand the virulence mechanisms of pathogenic E. coli,

which is key for developing preventive measures and treatments for colibacillosis in animals

and humans. Vaccines, probiotics, and antimicrobials targeting major virulence factors show

promise for reducing the substantial burden of this disease. There are various treatment and

vaccination approaches for avian colibacillosis which have been used by researchers. A brief

overview of an APEC infection is represented in figure 1.2.

The rise in the incidence of E. coli infections leads to unfeasible traditional antibiotic

treatment. This emphasizes the importance of alternative approaches to animal disease

prevention and treatment. One possible treatment for drug-resistant bacterial illnesses in

animals is phage therapy, which doesn't alter the normal gut flora. As a result, interest in the

field of biological disease control has grown rather than in the more traditional antibiotic

therapies. One such bio control method is the use of lytic bacteriophages that are unique to

pathogenic E. coli.

8
 Fig 1.2. Mechanism of APEC infection in chickens: The APEC infection in chickens
can progress to colibacillosis, causing multi-systemic effects and economic losses (Kathayat
et al., 2021).

Phage therapy has been used in a variety of fields, including agriculture, human and

veterinary medicine, and the treatment of E. coli infections in poultry. In the last two years

alone, numerous phage applications have been granted approval for use in the United States.

The most well-known ones are an anti-Escherichia coli wash, an anti-Salmonella wash, and

Listex, an organic anti-Listeria phage. These are used to treat live animals before they are

slaughtered (Durbas & Machnik, 2022).

1.4 Pathophysiology of Avian Colibacillosis

Colibacillosis in Chickens is characterized by the symptoms of lethargy, feather

dulling, diarrhea, and fecal matter stains around the cloaca. The clinical manifestation of

Avian Colibacillosis in birds is dependent on the immunological condition as a result of

feeding and housing conditions, as well as the pathogenicity of the bacteria. The more times

bacteria enters a bird's body, the more virulent it becomes. Autopsies of colibacillosis-dead

chickens indicate evidence of sepsis, including severe hemorrhages on the serous and mucous

membranes, internal organ enlargement, and spleen and liver enlargement (Ergashevich &

9
Salimovich, 2022). Several APEC virulence factors have been identified; however, no single

virulence feature has been established to distinguish an APEC from another. APEC virulence

characteristics recognized include adhesins, toxins, iron acquisition methods, invasins, and

plasmids. Temperature sensitive haemagglutinin (tsh), iron-scavenging systems such as iroN,

feoB, chuA, fyuA, ireA, irp2, iucD, and sitD, enhanced serum survival gene (iss), and

protectins are also mentioned (Newman et al., 2021).

Various risk factors can increase the chance of colibacillosis in broilers, such as

respiratory tract infections, and many environmental factors such as high ammonia and

carbon dioxide concentrations. Infectious E. coli belong to various polygenic groups as the

concentration of E. coli in the broiler houses is another determining factor of colibacillosis.

The lack of knowledge about virulence-associated genes is the major factor leading to high

mortality rates in the poultry sector (Azam et al., 2019). All age groups of birds and various

poultry production methods, such as broiler and commercial layers and breeders, are

susceptible to colibacillosis. Because E. coli's capacity to produce colibacillosis varies, its

function as a major or secondary pathogen that is triggered by different predisposing

circumstances is inconsistent and varies. For many years, the primary treatment for

colibacillosis has been antibiotics. On the other hand, the rise in antibiotic resistance has

made it necessary to find alternate treatments for colibacillosis in addition to putting a focus

on illness prevention (KOUTSIANOS et al., 2020). While a large number of strains are

benign, some can cause serious extraintestinal and intestinal illnesses. Extraintestinal

Pathogenic Escherichia coli strains are classified based on the host and the site of infection:

septicemia, respiratory tract infections (RTI), polyserositis, salpingitis, cellulitis, and

others. These are collectively referred to as colibacillosis; sepsis-associated E. coli (SEPEC),

neonatal meningitis E. coli (NMEC), and uropathogenic E. coli (UPEC). This uropathogenic

E. coli (UPEC) causes urinary tract infections (UTI), while avian pathogenic E. coli strains

10
(APEC) infect birds of all ages (Sattar et al., 2023). Avian colibacillosis, which affects the

poultry industry, can cause significant economic losses worth hundreds of millions of dollars

worldwide as well as high rates of morbidity and mortality in poultry (up to 20%)

(Abdelrahman et al., 2022).

1.5 Impact of Colibacillosis on Poultry Production in

Pakistan
The poultry industry is a very important part of the economy of Pakistan and

contributes 1.3 percent to the GDP of Pakistan. The poultry sector employs around 1.5

million people across Pakistan. The current investment in poultry is about 200 billion rupees.

Although the per capita meat consumption is very low compared to the developed countries.

There is still a lot of room to increase meat production at the domestic level (Soomro et al.,

2021).

Avian Colibacillosis is considered as one of the major culprit behind morbidity and

mortality in chickens. It also causes decreased meat production (up to 20%, 2% decline in

live weight, 2.7% deterioration in feed conversion ratio) lower hatching rates, and up to 43%

condemnation of carcasses at slaughter. E. coli is either the primary or secondary causative

agent and results in many related infections. These infections cause huge financial losses and

many diseases in the poultry sector such as respiratory tract infections and head swelling

syndrome (Shah et al., 2021). The acute form of avian colibacillosis results in septicemia and

its sub-acute form is characterized by pericarditis and yolk sac infection. However, there is

another condition that may cause yolk sac infection and that is fecal contamination which

may result in the transfer of E. coli to chickens through hatching.

According to a survey, colibacillosis affects at least 30% of commercial flocks in the

United States at any given time. It is typical to find Escherichia coli in the digestive tracts of

11
healthy birds and mammals. Typically, 10–20% of the avian intestine has Escherichia coli

that is pathogenic (APEC) and has a range of virulence factors (Amin et al., 2020). There

have been reports of a natural colibacillosis outbreak in young chickens, but the concurrent

illness in chicks of different age groups is quite intriguing, particularly in light of the

diversity of serogroups with varying pathogenicity capacities.

The economy of Pakistan depends heavily on the poultry industry, however

Escherichia coli infections have a detrimental effect on the poultry business of the country.

Significant morbidity and mortality from APEC contribute to multimillion-dollar losses for

the global poultry business in all its forms (Ganaie et al., 2021). There is, however, no

information on the relationship between different pathologies observed in spontaneous cases

of colibacillosis and Escherichia coli serogroups (Apostolakos et al., 2021). Sequence types

(STs) based on phylogenetic subgroups can characterize the strong population structure of

Escherichia coli. MLST, however, can be costly and time-consuming. Recently, effective

molecular typing instruments have been created and verified. Allele-specific PCR is one such

method that has demonstrated consistent effectiveness in identifying STs associated with

different E. coli subgroups. Numerous epidemiologic analyses have been superseded by this

method. Several extra-intestinal illnesses seem to favorably contain some of these groupings

(Usman et al., 2023). Southeast Asian nations are major exporters of chicken goods, making

poultry a vital regional sector. However, this industry is heavily impacted financially by

infectious diseases like colibacillosis (Rafique et al., 2020).

1.6 Bacteriophages as Therapeutics for Colibacillosis

Bacteriophages, often known as phages, can be used as alternatives in place of

antibiotics and vaccinations. Frederick Twort and Felix d'Herelle reported the discoveries of

bacteriophages in 1915 and 1917, respectively. Following that, there have been suggestions

12
that treating bacterial illnesses could involve administering these viruses. Phage treatment

had encouraging first results, but its use became limited because penicillin was discovered in

1928 and antibiotics were produced on a large scale in 1940 (Chanishvili, 2012). Numerous

investigations were carried out to evaluate the effectiveness of lytic phages in treating

bacterial illnesses before the discovery of antibiotics. Phages are superior to antibiotics in

several ways. Phages are naturally occurring agents that eliminate bacteria and are regarded

as the most prevalent microbial species. There are around 1031 phage virions that exist on

planet earth (Abbas et al., 2022). Phage therapy also has some additional benefits over

antibiotics since phages are easily and affordably produced, and they are readily available in

the environment. Furthermore, unlike antibiotics, they are specific to bacterial targets and do

not negatively affect the natural microbiota. However, bacteriophage research was

discontinued, except in the former Soviet Union and Eastern Europe.

Since bacteriophages are found in all environments and are thought to be ten times

more numerous than all known bacteria, they are excellent choices for the treatment of

infectious diseases. This mechanism of action does not kill the commensal gut flora.

Bacteriophages self-replicate throughout therapy, therefore frequent application is not

necessary. Phage titers decrease as a result of their incapacity to adhere to and proliferate in

eukaryotic cells, and this is correlated with a markedly reduced quantity of harmful bacteria

infecting the organism. Since proteins and nucleic acids make up the majority of phages, their

non-toxicity is another crucial feature. Because phages are so specific to a particular bacterial

strain, bacteriophage therapy is becoming more and more popular (Naureen et al., 2020).

Bacteriophages specific for E. coli have been isolated from environmental and clinical

sources. E. coli phages are classified into virulent phages and temperate phages based on their

replicative lifestyle. Virulent phages like T4 and T7 solely undergo the lytic life cycle upon

13
infecting E. coli, wherein the phage hijacks bacterial biosynthetic machinery to mass produce

viral components. This culminates in cell lysis and death, releasing a burst of progeny virions

that reinitiate infection of other host cells (Juškauskas et al., 2022).

Temperate phages like lambda demonstrate either lytic or lysogenic life cycles. In

lysogeny, phage DNA is integrated into the host chromosome in the form of a prophage that

replicates passively with the host DNA. The Lambda repressor ci prevents transcription of

lytic genes in the prophage state. Stress can induce spontaneous prophage excision and

initiation of the lytic cycle. E. coli phages include T-even phages T2, T4 and T6, lambdoid

phages λ and Ø80, and ssRNA phages like MS2 and Qβ (Golding et al., 2019).

Bacteriophages have several appealing advantages over antibiotics when utilized for

biocontrol. They have a better host specificity than other antimicrobials, which minimizes the

risk of damage to non-target microbiota, and phage cocktails allow for the treatment of

different strains of the same pathogen by enhancing the effectiveness. Bacteriophages may be

administered once and be sufficient for treatment unlike other antibiotic treatments that may

need multiple daily doses to completely eradicate bacteria. Furthermore, because

bacteriophages can be administered orally or through food or drink, the technique of applying

them is simple. Despite being widely distributed in the environment, not all bacteriophages

are appropriate for the in vivo prevention and treatment of illnesses (Romero-Calle et al.,

2019).

Endogenous bacteriophages may not be able to control bacterial infection because of

changes in bacteriophage functions during infection brought on by things like an acidic

environment in the gastrointestinal tract, an elevated body temperature, and the presence of

naturally occurring anti bacteriophage antibodies (Rahimi-Midani et al., 2021). Furthermore,

an improper bacteriophage dosage that is applying a phage at a high infection multiplication

14
may cause a condition called "lysis from without." Under this situation, a huge number of

phages kill the bacteria, causing the host bacteria to lyse prematurely without releasing free

phage progeny for the subsequent infection cycle. In these circumstances, the phage may

have damaged the host cell membrane, causing the bacteria to lyse instead of the infection.

Therefore, prior to determining the phage's effectiveness, in vivo research on isolated

bacteriophages is necessary (Hitchcock, 2023).

1.7 Cloning of endolysins of Bacteriophages

Phage therapy has a lot of potential applications in agriculture, veterinary medicine,

and human medicine. Despite this, low host specificity and the potential for phage resistance

limit the use of phages as antibacterial agents. On the other hand, lytic enzymes that come

from phages have a lot of benefits. The use of phage lytic enzymes does not result in

resistance development aside from a wider substrate range and distinct physicochemical

properties. The most often used lytic enzymes are endolysin and holin, which are two key

phage proteins involved in host lysis. Endolysins are lytic enzymes encoded by

bacteriophages. After phage replication, endolysins can hydrolyze bacterial peptidoglycan to

release additional phage particles from host cells. Endolysins are enzymes produced by

bacteriophages during the viral replication cycle to degrade the bacterial cell wall and release

new virions (Nazir et al., 2023). Endolysins are classified into five groups according to their

specificity: endopeptidases, amidases, glucosidases, muramidases, and carboxypeptidases (Lu

et al., 2020).

Cloning of bacteriophage endolysins can be an effective strategy for developing novel

antimicrobial agents against drug resistant bacteria. In recent years, there has been growing

interest in cloning and expressing endolysins as a novel class of antimicrobials. Endolysins

have a very specific spectrum of activity and only target the bacteria they have evolved to

infect as compared to traditional antibiotics, which makes resistance development less likely

15
(Murray et al., 2021). Apart from endolysin, holin is another essential hydrophobic protein

that regulates host lysis. It collaborates with endolysin to produce timely and rapid host cell

lysis. Because Holin is able to regulate the timing of phage infection cycles and lyse host

cells at the optimal time, it is also referred to as "the clock to control phage infection".

Holins have several features, but they do not share sequence similarities. The majority

of holins are encoded by the gene next to the endolysin gene. At least one hydrophobic

transmembrane domain is present in every Holin (Abeysekera et al., 2022). The C-terminal

domain of holins is hydrophilic and highly charged. By looking for these characteristics,

probable Holins can be predicted. Other than that, Holins have been investigated in

experimental model systems as possible treatments. Various pathogen phage endolysins and

holins have been employed as antibacterial agents to treat antibiotic-resistant bacteria. The

particles of the assembled phage burst out via a Holin-induced gap that is developed between

the cell wall's damaged interior and inner membrane. The recombinant endolysins may harm

them from the outside, serving as antimicrobials by breaking down the cell membrane of

bacteria (Rahman et al., 2021).

The genes that produce endolysins were found and successfully cloned from three

different Escherichia coli bacteriophages: PBEC30, PBEC56, and 10-24 (13) (Jeong et al.,

2023). The putative antimicrobial peptide (AMP)-like C-terminal alpha helix structures with

amphipathic properties were predicted using the three endolysins. Hexahistidine-tagged

versions of each gene were cloned, produced, and the resulting proteins were purified and

described. A range of Gram-negative bacteria, such as and Klebsiella pneumonia,

Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter baumannii were susceptible to

the antibacterial properties of the refined endolysins. Their N-terminal was fused with the

antimicrobial peptide cecropin A to increase their antibacterial activity. As low as 4 μg/mL

could be the minimum inhibitory concentration (MIC) for the targeted strain. The enzymatic

16
activity of the endolysins remained constant between 4 and 65 °C and were unaffected by pH

variations between 5 and 10. Galleria melonella infection models were used to illustrate the

three endolysins' in vivo efficacies.

In the current study, the endolysin and Holin gene from the isolated bacteriophages

were cloned and their expressions were optimized. The recombinant endolysin and holin

proteins will be purified through column chromatography. The therapeutic potential of

isolated bacteriophages and purified holin and endolysin recombinant proteins were checked

by double layer agar method.

1.8 Control and Prevention of Colibacillosis

The control and prevention of colibacillosis can be difficult, and management-focused

measures have had mixed results because E. coli are a normal component of bird intestinal

flora. The two strategies for preventing colibacillosis include Biosecurity, which controls

personnel access, and bird mobility, which restricts the entry of pathogenic E. coli and lowers

flock exposure. It is also possible to restrict the growth of pathogens in the flock and reduce

the quantity of E. coli in the water and feed by ensuring that the environment is sufficiently

clean and that the temperature, humidity, and ventilation are optimal. More importantly,

colibacillosis risk can be decreased by shielding flocks from other viral or bacterial diseases

that results in weakened host resistance. For APEC, antibiotics are also utilized, either in

flocks where a portion of the birds are already sick in order to prevent additional illness or

death (metaphylaxis) or in flocks where the birds are healthy but may become sick in the

future (prophylaxis). Due to virulence and resistance plasmids, highly virulent and resistant

strains of colibacillosis have emerged, posing challenges to current efforts in the prevention

and control of the disease (Nolan et al., 2013).

The World Health Organization (also known as the WHO) and the World

Organization for Animal Health (OIE) have both issued guidelines on responsible

17
consumption of antimicrobials in the face of the threat of antimicrobial resistance. It is widely

accepted that in order to lower the risk of antimicrobial resistance, antibiotics should be used

carefully in both humans and animals. Veterinarians and poultry experts must have access to

impartial, reliable information about the effectiveness of antibiotics in preventing and

controlling colibacillosis in broilers if they are to minimize the use of antibiotics. By

comparing the advantages and disadvantages of different antibiotics, practitioners can make

well-informed decisions and choose the most suitable and successful preventive measures or

treatments (Sargeant et al., 2019).

1.9 Alternative treatment options for Colibacillosis

Antibiotics are often used to treat colibacillosis, but there are some alternative

treatment options that may be effective as well. The antimicrobial resistance has the potential

to severely impact the management and treatment of infectious diseases in both humans and

animals. When using non-antibiotic treatments to control APEC, the application of an

alternate rational approach is essential. Tests have been conducted on probiotics,

bacteriophages, growth inhibitors, virulence, innate immune stimulants, and antimicrobial

peptides (Kathayat et al., 2021). Biofilms also hinders the ability of conventional antibiotics

to enter cells, reducing their susceptibility to treatment. Therefore, there is a critical need to

research alternative treatments to address infections caused by isolates of E. coli biofilm

development (Gebreyohannes et al., 2019).

Several studies have indicated that probiotics can help prevent and treat diarrhea

caused by E. coli. Specific probiotic strains like Lactobacillus rhamnosus GG and

Saccharomyces boulardii have been shown to shorten the duration of diarrhea, reduce stool

frequency, and lessen intestinal inflammation. Probiotics are available as supplements in

capsule, powder, or liquid forms. They can be used alongside antibiotics or on their own

(Săsăran et al., 2023). Some other alternative or complementary options for colibacillosis

18
treatment being explored include zinc supplements, probiotic enemas, fecal microbiota

transplantation, and bacteriophage therapy. More research is needed to confirm safety and

efficacy. Integrative approaches that combine conventional care with alternative therapies

tailored to the patient also show promise for improved outcomes. One of the best methods for

treating drug-resistant bacterial infections in animals without altering their natural gut flora is

phage therapy. Numerous characteristics of bacteriophages make them potentially attractive

treatment agents for bacterial diseases. Among them include the outstanding efficacy and

specificity in lysing dangerous bacteria that are the target (Nabil et al., 2018). The strategy

used during the phage-bacterium interaction was based on the breakdown of the extracellular

polymeric substance present in the biofilm by the production of polysaccharide de-

polymerases. This process allowed the phage to enter and encase the bacterial cells, and

causing their lysis

1.10 Significance of the Study

Colibacillosis has a devastating impact on the economy of Pakistan with the mortality

rate varying from 20% to 40%. As a result, interest in the subject of biological disease control

has grown rather than in the more traditional antibiotic therapy. Since lytic bacteriophages

have a number of benefits over antibiotics, using them as therapeutic agents is one such bio-

control alternative. Only the pathogens of interest are targeted by phages, leaving the system's

regular microbiota intact. Their mode of action is completely different from that of any

antibiotic currently in use, and they have also demonstrated efficacy against bacteria that are

resistant to several antibiotics. The effects of phage therapy are entirely confined at the

infection site, while antibiotics may not always focus at the initial site of infection (Azam et

al., 2019).

The significance of this work is the potential of bacteriophages and their derivatives

as alternatives to traditional antibiotics for treating multiple drug resistant E. coli infections.

19
Phage therapy represents a promising strategy to combat colibacillosis and reduce financial

losses and mortality on poultry farms with increasing antibiotic resistance and a decline in

new antibiotics under development. The characterization and efficacy testing in this study

will provide the necessary evidence to position bacteriophages as viable and potent

therapeutics. There is evidence that Bacteriophages may penetrate the blood-brain barrier

and enter tissues with inadequate vascularization. In addition to being helpful in the isolation

and characterization of new bacteriophages targeting MDR E. coli, this work included

successful experiments to manage colibacillosis infection in Pakistan's Poultry industry. This

study will provide an innovative strategy and course of action for treating MDR bacteria by

screening phages that are active against MDR. This will also contribute to our understanding

of the potential applications of phages (mono-phages and cocktails) or recombinant proteins

(holins and endolyisn) generated by phages as therapeutic agents.

1.11 Aims and objectives

The key objectives of this research were the isolation and characterization of

bacteriophages against Escherichia coli O119 which was isolated from the chickens infected

with Avian Colibacillosis. The next steps were the evaluation of their efficacy in controlling

infections, and their molecular characterization through next-generation sequencing,

amplification, and cloning of endolysin and holin genes. The focus of the study was the

isolation of bacteriophages capable of infecting and lysing E. coli O119 from environmental

sources such as sewage. Promising lytic phages were then characterized in terms of host

range, efficiency of plating, stability, and burst size.

Another focus of the study was assessing the efficacy of the isolated bacteriophages

alone or in cocktails to control E. coli O119 infections in vitro and in vivo using animal

infection models. This will demonstrate their potential for phage therapy against

colibacillosis in poultry farms. This study also focused on the molecular characterization of

20
the most promising bacteriophages through whole genome sequencing and bioinformatic

analysis. The potent endolysin and holin genes encoded in these phage genomes were then

amplified, cloned, and the proteins were also purified. Moreover, testing these enzymes

against target bacteria was used to evaluate their potential as alternative antimicrobials.

The main objective of this study was to isolate and characterize the bacteriophages

against E. coli causing poultry colibacillosis. Here is a brief outline of the objectives of this

study, which are summarized as under.

I. Isolation, biochemical and molecular characterization of avian pathogenic E.coli.

II. Isolation and characterization of lytic phages against poultry MDR E.coli bacteria.

III. Molecular characterization of bacteriophage by full genome sequencing using next

generation sequencing.

IV. Measuring and accessing the effectiveness of phage cocktails.

V. Cloning and expression of endolysin and Holin genes of lytic phage isolates.

VI. To study therapeutic potential of bacteriophages cocktails and their gene products (Holin

and endolysin) against MDR E. coli.

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