RESEARCH PROPOSAL

DEVELOPMENT OF A BACTERIOPHAGE COCKTAIL AGAINST AEROMONAS SPECIES

Course name- MSc Biotechnology (2022-2023)

Subject- Advance scientific writing
Paper- PSBT 404 – Unit II
Faculty- Dr. Archana Rath
Name- Uditi Chetan Jaiswal
Date- 13/04/2023
 DEVELOPMENT OF A BACTERIOPHAGE COCKTAIL AGAINST AEROMONAS SPECIES

ABSTRACT
In this dominating era of multi drug resistant bacteria, intensive study and research is being
carried out on bacteriophages which has the capability to act as a potential therapeutic
agent popularly known as phage therapy. Bacteriophages (phages/viruses) need host
bacteria to replicate and propagate while some pathogenic bacteria like Aeromonas
salmonicida which is a fish pathogen that infects its host salmonid cause furunculosis
disease. The post treatment achievement using mix phage might show a high survival rate
as compared to a single phage. Thus the research aims to design a mixture or a cocktail of
bacteriophage which can control the infection of Aeromonas species in salmonids and
reduce the global economic loss occurring in the field of aquaculture. As per the research
done earlier, lytic phage has been considered as an effective bactericidal agent.
KEYWORDS: phage therapy, furunculosis, fish pathogen, lytic, aeromonas, antimicrobial
resistance
INTRODUCTION
The genus Aeromonas (phylum, Proteobacteria; class, γ-Proteobacteria; order,
Aeromonadales; and family, Aeromonadaceae) comprises a collection of ubiquitous gram-
negative bacilli that are widespread in aquatic environments. It is a waterborne pathogenic
fish bacterium and a causative agent to cause disease named as furunculosis in salmonids
mainly. The disease furunculosis is characterized by the furuncle or boil like swellings on the
skin of the fishes. Intensive uses of antibiotics in order to treat and prevent the furunculosis
in salmonids lead to the development of multi drug or multi antibiotics resistance in
Aeromonas salmonicida and thus there is a need to find an alternative or a solution to
control the spread and development of the infection.
Bacteriophage is a virus that particularly infects its host bacteria and is dependent on the
host’s replication machinery for its own replication, propagation and survival.
Bacteriophages ranges from the size of 25-200nm. Bacteriophages are of two types on the
basis of its life cycle viz. temperate/ lysogenic phage and virulent/ lytic phage. Temperate
phage enters in the bacterial chromosome and reproduce without killing its host whereas
virulent phages insert in its host bacteria and cause lysis. The ability of a bacteriophage to
specifically infect various types or strains or speices of bacteria is termed as the host range
of that bacteriophage. It is obvious to some extent that a single type of phage will have a
narrow host range while a mixture or a cocktail of bacteriophage will have a broad host
range i.e. the ability of infect varied number of bacterial species and strains.
OBJECTIVES
The goal of this research is to design and develop a cocktail of bacteriophage against
Aeromonas species causing disease in fishes. This cocktail should have the ability to kill the
Aeromonas species in the aquatic environment and the fishes should grow healthy. The
research aims to prevent the loss that occurs in aquaculture due to the continuous misuse
and overuse of antibiotics.
METHODOLOGY
1. Collection of the sample from the site
Sewage water sample and water sample from the aquatic environments will be
collected to isolate the bacteriophages and its host bacteria. The strains should also
be used from the repositories. All strains to be stored at -80 0C. 16S rRNA gene
sequencing or complete genome sequencing should be done of all strains.
2. Isolation and purification of bacteriophage
The sewage water is generally considered to be a rich source of the bacteriophages
due to presence of their host. Collect all these samples (50 ml each) in the sterile
falcon tubes separately. Add chloroform to all the samples at 0.1% (v/v) and keep on
orbital shaker at 30 °C for 2 h. Centrifuge at 8000 g for 25 min to sediment bacterial
debris. Pass the supernatant through sterile 0.45 µm pore size syringe filters and
then again pass through 0.22 µm pore size syringe. Collect the filtrates separately in
sterile falcon tubes and store at 4 °C. Use these processed water samples as the
source of the bacteriophages.
3. Enrichment of Aeromonas phages
Aeromonas salmonicida to be used as a host for enrichment of Aeromonas phages
from the processed water samples by the method described previously. Twenty-five
millilitres of processed water samples (25 ml) should be mixed separately with equal
volume of 5 X TSB supplemented with 1 mM CaCl2 and inoculate with overnight (o/n)
grown culture of A. salmonicida separately in Erlenmeyer flasks. In the control flask,
the sterile distilled water should be used in place of processed water sample. These
flasks should be incubated o/n at 30 °C on orbital shaker at 180 rpm. Following
incubation, the flasks have to be observed for the clearance. The contents of the
flasks have to be transferred to sterile tube (volume = 50 ml) separately. The host
bacteria should be settled by centrifugation (10000 g) for 20 min. The supernatant to
be filtered through sterile 0.45 µm pore size Ministart syringe filters and then again
 pass through 0.22 µm pore size Ministart syringe filters. The filtrate to be designated
as cell-free enrichment lysate (CFEL).
4. Host range analysis
Spot assay- The host range of the phage in the phage stock can be studied by spot assay
where 10 µl of each CFEL can be spot inoculated on the lawn of different bacteria.
Following the o/n (16-18 hrs) incubation, the plates to be observed for the zone of lysis.
5. SEM and TEM
Scanning electron microscopy and Transmission electron microscopy to be
performed in order to understand the structure of the bacteriophage and identify
the family and type of phage. GenBank databases to be used to identify the
phylogenetic analysis
6. Characterization of bacteriophage
Effect of temperature, pH and other factors can be determined on bacteriophage by
incubating the bacteria and phage for specific time interval using dilution and agar
overlay method.
7. Adsorption curve
Phage adsorption curves can be generated by adding the phage suspension the the culture
at multiplicity of infection (MOI) and incubate mixtures at 30 0C. Next, 100 µl of the mixture
can be collected at specific time points and dilute immediately with 900 µl of cold LB
medium. After centrifugation (12,000 × g, 2 min), the supernatants to be titrated to
determine the number of un-adsorbed phages.
8. Bacteriophage killing curve and In Vitro phage cocktail design
Bacterial inactivation can be determined individually for each phage and then various phages
can be combined to determine its effect on the bacteria
TIMELINE
Category 1- Literature review and designing the research
Category 2- Isolation, enrichment and host range analysis of bacteriophage
Category 3- Characterization
Category 4- Combining various isolated phages and determination of bacterial inactivation
 Timeline

4
3

1.5

Category 1 Category 2 Category 3 Category 4

Months

BUDGET

SR. PARTICULARS AMOUNT TOTAL JUSTIFICATION

NO.

1 Senior scientific officer 60,000/- 60,000/- In order to guide, observe and tackle the
challenges that will come up during the
research period. The officer should have
dealt and completed publishing research
work related to bacteriophage.

2 JRF- 3 40,000/- 1,20,000/- In order to perform the experiments like

each characterization and genome sequencing
that will go hand in hand. The JRF should
have the basic knowledge of
bacteriophage and efficient in
microbiology practices.

3 Equipments- Shaker incubator is needed for the

Shaker incubator at 3 lacs cultures to grow. Biosafety cabinet will be
300C 4.9 lacs needed in order to perform the plaque
Biosafety cabinet class 2 10 lacs 37.4 lacs assay and various all other
Refrigerator at -800C 5 lacs microbiological experiments. PCR
machine to be provided for the detection
 PCR machine 14.5 lacs of genes present in phage and host
Nanodrop bacteria. Nanodrop photospectrometer
photospectrometer needed to determine the DNA quantified
adter DNA extraction.

4 Others 1 lac 1 lac For performing all the experiments

Glasswares
Reagents and chemicals
Media

OVERALL OUTCOME OF THE PROJECT

1. To design an efficient cocktail of bacteriophage i.e. an alternative antimicrobial
agent against multidrug-resistant Aeromonas salmonicida
2. To benefit the aquaculture industry using the phage therapy as the biocontrol
mechanism.

REFERENCES
1. Yu, Huabo & Zhang, Liang & Feng, Chao & Chi, Teng & Qi, Yanling & Raza, Sayed
Haidar Abbas & Gao, Na & Jia, Kaixiang & Zhang, Yang & Fan, Ruining & Cai, Ruopeng
& Qian, Aidong & Li, Ying & Sun, Wuwen & Shan, Xiaofeng & Liu, Ning & Zhang, Lei.
(2021). A phage cocktail in controlling phage resistance development in multidrug
resistant Aeromonas hydrophila with great therapeutic potential. Microbial
Pathogenesis. 162. 105374. 10.1016/j.micpath.2021.105374.
2. Muhsin Jamal, Sayed M. A. U. S. Bukhari, Saadia Andleeb, Muhammad Ali, Sana Raza,
Muhammad A. Nawaz, (2018) Bacteriophages: an overview of the control strategies
against multiple bacterial infections in different fields 10.1002/jobm.201800412
3. Ling Chen1 , Shengjian Yuan1 , Quan Liu1,2 , Guoqin Mai1 , Jinfang Yang3 , Deng Deng3 ,
Bingzhao Zhang1 , Chenli Liu1 and Yingfei Ma1 (2018) In Vitro Design and Evaluation of
Phage Cocktails Against Aeromonas salmonicida 10.3389/fmicb.2018.01476