Comparative anti-bacterial efficacy of Salmonella enterica bacteriophage and antibiotics

Comparative anti-bacterial efficacy of Salmonella enterica bacteriophage and antibiotics (Ciprofloxacin and Gemifloxacin) against Salmonella infection
A Thesis Proposal Submitted to the
Department of Biology
College of Arts and Sciences
University of the Philippines Manila
Submitted in Partial Fulfillment of the Requirements in
Biology 199 – Research Methodology
Submitted by:
Kiana Marie G. Canete
2015-04424
BS Biology
CHAPTER 1
INTRODUCTION
Background of the Study
The increasing number of microorganisms that acquire or develop antimicrobial resistance (AMR) which are referred as “superbugs” serves as a serious threat to the global public health (WHO, 2018). According to the World Health Organization (WHO), the success of the treatment and prevention of certain diseases caused by these so-called “superbugs” are compromised due to the absence of effective antibiotics. The AMR is present in every country as stated by the WHO but the developing countries are in much greater risk compared to the first-world countries due to the limited access to new treatments and prevention techniques against the “superbugs”.

One of the microorganisms that developed antimicrobial resistance is Salmonella spp., a gram-negative, rod shaped bacillus, one of the most common causes of food poisoning which can be found in contaminated water or foods such as meat, poultry, and eggs (Nordqvist, 2017). This bacterium causes the following symptoms to appear 12 to 72 hours after infection: abdominal cramps, diarrhea, vomiting which usually goes away on its own however, in severe cases hospital treatment is needed, Nordqvist added.

According to the Centers for Disease Control (CDC), the resistance of Salmonella strains against antibiotics continue to increase with four primary serotypes being responsible for the resistance – Enteritidis, Typhimurium, Newport, and Heidelberg (Dall, 2016). The Salmonella antibiotic resistance continue to threaten the public health worldwide especially in the developing countries such as the Philippines. A study conducted by Kamela Charmaine Ng and Windell Rivera, students of University of the Philippines, revealed that the strains of Salmonella enterica which were acquired from tonsil and small intestine tissues of swine slaughtered in Metro Manila are multidrug-resistant (MDR) which may cause a huge challenge in the treatment of the disease and play a significant role in preventing the spread of these MDR strains among hogs and humans (Philippine Council for Health Research Development, 2014).
A potential alternative way of treating Salmonella infection is the use of bacteriophages. This treatment is known as bacteriophage therapy which uses phages or bacterial viruses to infect and cause lysis to the target bacteria (Colindres et al., 2011). The bacteriophage that will be isolated from the Salmonella enterica will be used and introduced to the infected mice to test its effectivity against the infection.

Statement of the Problem
Can Salmonella enterica bacteriophage be used as a better treatment for Salmonella infection compared to Fluoroquinolones specifically Ciprofloxacin and Gemifloxacin?
Objectives of the Study
In order to effectively study the significance and capabilities of the bacteriophage of Salmonella enterica the following objectives must be met:
Specifically, the study plans to:
successfully isolate a bacteriophage from Salmonella enterica.

test the activity of the isolated S. enterica phage by knowing the average plaque size produced.

determine if there is a difference in the lethality of different concentrations of S. enterica phage used as a treatment for Salmonella.

determine whether S. enterica phage or Fluoroquinolones – Ciprofloxacin and Gemifloxacin is more effective in treating Salmonella infection by comparing the amount of bacteria present after the treatment.

Hypothesis:
Ho : There is no significant difference between the efficacy of S. enterica bacteriophage and antibiotics (Ciprofloxacin and Gemifloxacin) in treating Salmonella infection.

Ha: There is a significant difference between the efficacy of S. enterica bacteriophage and antibiotics (Ciprofloxacin and Gemifloxacin) in treating Salmonella infection.

Significance of the Study
The study is deemed significant since it would be able to provide more knowledge in improving the healthcare system specifically in treating and controlling the Salmonella infection especially in the occurrence of antibiotic resistance. Aside from adding knowledge in the medicinal aspect, the study would also be able to give a better understanding on how Salmonella spp. infects its host and how to prevent them from spreading inside the host which can help in the agriculture industry.

Through this study, additional knowledge about bacteriophage therapy may be gathered. Different serotype of the Salmonella species can be tested if it is possible to treat certain diseases. Alternative medicine would also be discovered since the fluoroquinolones have side effects in the human body.

Scope and Limitations of the Study
The study will be focusing on the Salmonella enterica species which causes Salmonella infection. The bacteriophage that will be isolated will also come from the species S. enterica that will be collected from the meat infected by Salmonella enterica strain collected from the markets here in Metro Manila. The antibiotic that will be used to treat the disease and be compared to the phage is the fluoroquinolone antibiotic – Ciprofloxacin and Gemifloxacin that will be acquired from the authorities.
CHAPTER 2
REVIEW OF RELATED LITERATURE
Salmonella
Salmonella is a gram-negative, rod shaped bacillus that has caused illness for over 125 years and was discovered by Dr. Salmon, an American scientist. This bacterium is one of the most common causes of food poisoning which can be found in contaminated water or foods such as meat, poultry, and eggs (Nordqvist, 2017). According to the Centers for Disease Control and Prevention (CDC), people infected with Salmonella experience the following symptoms 12 to 72 hours after infection: diarrhea, fever, and abdominal cramps which usually lasts for about 4 to 7 days. Usually, people with this illness can recover without any treatment however, during severe cases the patient needs hospital treatment, stated by the CDC. According to the Public Health Agency of Canada, all Salmonella species (enterica and bongori) can infect humans.

Salmonella enterica
Samonella enterica, formerly known as Salmonella choleraesius is one of the two Salmonella species. A motile, aerobic to facultatively anaerobic, nonspore-forming bacterium and is a common inhabitant of the gastrointestinal tract of ruminants which makes it a major cause of food-borne illness in humans (Public Health Agency of Canada, 2010). According to the Public Health Agency of Canada, this Salmonella species can cause four different clinical manifestations: gastroenteritis, bacteremia, enteric fever, and an asymptomatic carrier state. The treatment that will be applied for these diseases depends on the clinical symptoms that the patient has, as stated by the Public Health Agency of Canada.
Fluoroquinolone – Ciprofloxacin and Gemifloxacin
Fluoroquinolones are antibacterial agents that are used widely as treatment for respiratory and urinary tract infections by exhibiting concentration-dependent bactericidal activity through the inhibition of the activity of DNA gyrase and topoisomerase which are enzymes essential for bacterial DNA replication (Schlecht & Bruno, 2015). This family of antibacterial agents that includes Ciprofloxacin and Gemifloxacin are active against a wide range of aerobic gram-positive and gram-negative organisms which can be used to treat bacterial gastroenteritis and typhoid fever – caused by Salmonella.
However, recent studies show that Salmonella enterica is developing resistance against Ciprofloxacin which is causing global concern. According to a study conducted by Raveendran et al (2008), there are serotypes of S. enterica which is resistant to ciprofloxacin while others remain susceptible to it. The researchers concluded that ciprofloxacin can no longer be considered as treatment for Salmonella infections.

Fortunately, there are still no studies about the development of resistance of Salmonella against Gemifloxacin.

Bacteriophage therapy
A potential alternative way of treating tetanus is the use of bacteriophages. This treatment is known as bacteriophage therapy which uses phages or bacterial viruses to infect and cause lysis to the target bacteria (Colindres et al., 2011). There are already studies conducted here in the Philippines regarding bacteriophage therapy wherein the researchers were able to isolate and compare the performance of the phages and the antibiotics used for a certain disease such as burn wound infection caused by Pseudomonas aeruginosa. An example of a successful isolation of a phage in the country is the MCR-10 which according to Colindres is the first Klebsiella pneumoniae bacteriophage. The study conducted by the group of Colindres showed that the phages of P. aeruginosa and K. pneumoniae have great potential for therapy after showing comparable results to antibiotics. Meanwhile, this study opened an opportunity for another research about bacteriophages which focused on the performance of P. aeruginosa phage against burn wound infection. The results of the experiment showed that MB-08, the P. aeruginosa phage, has a therapeutic significance in treating burn wound infections after showing a significant reduction in the number of bacteria present in the rats compared to the rats treated with antibiotics (Abengaña et al , 2012). These researches will serve as a guide for the researchers in testing the potential of the S. enterica phage against Salmonella infection and determining the differences between the performance of the phage and the known antibiotics used to treat the infection.

The mentioned studies open up new possibilities and opportunities in the potential of bacteriophage therapy as an alternative for antibiotics. According to Lin et al. (2017), the renewed interest in practicing the phage therapy even though being around for almost a century is due to the declining effectiveness of antibiotics in treating diseases. Further studies should be done about the effectiveness of page therapies to provide new knowledge about these phages which can be used in the advancement of the medical field especially here in the Philippines.

Conceptual Framework
Independent variables:
the dosage of the bacteriophage that will be introduced
the antibiotics that will be used as treatment
Dependent variables:
the lethality of the S. enterica at different concentrations
the average number of bacteria present in the blood after the treatment
the average size of plaques that will grow
Definition of Terms
Culture – the organisms that grow in the culture medium after being inoculated
Therapy – treatment
CHAPTER 3
MATERIALS AND METHODS
Experimental Design
The Salmonella enterica will be isolated from the infected meat samples which will be gathered from the Metro Manila markets. The isolated S. enterica samples will be used to produce the S. enterica bacteriophage which will later on be used as a treatment for the mice that will be infected with S. enterica. The antibiotic fluoroquinolone specifically Ciprofloxacin will also be used as a treatment for the infected mice. The efficacy of the phage and the antibiotic will be compared by determining the amount of bacteria present in the blood of the mice after the treatment.
Data Collection and Procedure
Bacterial organism
The Salmonella enterica will be isolated from the meat samples specifically infected swines in markets around Metro Manila which will be maintained in Typtic Soy Agar (TSA) slants at 4º C.
Phage isolation and selection
The meat samples will be screened for the presence of phages by a spot test. A loopful of S. enterica will be inoculated into 10 mL of Tryptic Soy Broth (TSB) which will be incubated for 12 h at 37º C. The resulting broth culture will be used as the host organism. The tubes containing 2ml of soft agar will be melted in a water bath and will be kept at 42º C. After that, 0.5 mL of the host organism and 0.1 mL of the phage filtrates will be added to the tube containing soft agar. The contents of the tube will be poured onto the surface of TSA plates. After the soft agar layer solidified, the plates will be incubated at 37º C for 24h. Plates that will exhibit plaque formation will be selected to be used as treatment for mice that will be infected with S. enterica.
Enumeration of bacteriophages
The enumeration of the bacteriophages will be carried out by making serial dilutions of the S. enterica phage filtrates. Each dilution of phage suspension will be subjected to the agar layer method that was described earlier. Plates will be incubated at 37º C for 24h. After incubation, the number of plaques will be counted on the appropriate dilutions. The dilution containing 108 and 1010 CFU/mL will be used for the animal model of the experiment.

Animals (Mice)
Sprague-Dawley female rats (7-10 weeks old) will be obtained from Plegaria Biological & Zoological Supply, Binangonan, Rizal. All animals will be given food and water ad libitum. The animal study will be conducted following protocols approved by the Institutional Animal Care and Use Committee. All the experiment that will be conducted is in in triplicate.

Introduction of the Bacteria and the phage to the mice
The isolated S. enterica sample will be introduced to the mice intravenously. After allowing the microorganism to multiply inside the mice, the phages (107 plague-forming units PFUs in 100 uL of saline) will also be inoculated intravenously,
Antibiotic and bacteriophage treatment
The efficacy of phage and antibiotics will be evaluated by using six groups of rats with three rats each. S. enterica will be introduced in all groups except for group 1. In group 1, rats were left without introduction of S. enterica and will serve as negative controls. In group 2, all infected rats will be left untreated which will serve as positive controls. In groups 3 and 4, rats will be infected and will be treated with Ciprofloxacin with different dosages, respectively. In groups 5 and 6, infected rats will be treated with different concentrations of the phage – 108 and 1010 PFU/mL, respectively.
Enumeration of bacteria present in blood
After 96 h post-infection, blood samples will be collected from each rat through tail clipping. A clean pair of sharp surgical scissors will be used and not more than 3 millimeters of the tail be snipped. Two drops of blood, approximately 100 µL, from the tail of each rat will be allowed to drop on duplicate plates, and then spread plate technique will be performed. All plates will be incubated for 24 h at 37ºC and colonies one each will be counted.

Data Processing and Analysis
Statistical Method
The statistical method that will be used in order to process, analyze, and interpret the acquired data is the Student’s t test for direct mean comparison using the GraphPad Software. Difference with P£0.05 will be considered as statistically significant.

The methodology were based on the study conducted by Abengaña et al (2012) entitled Comparative treatment of Pseudomonas aeruginosa burn wound infection using bacteriophage MB08 and antibiotics.

CHAPTER 4
RESULTS
9525025912100The average size of the plaques will be recorded and pictures will also be inserted.

95742365358500 Figure 1. Lethality of S. enterica at dose 108 CFU/ mL in the mice. A score of 5 indicates normal health, while 0 indicates death.

Figure 2. Lethality of S. enterica at dose 1010 CFU/ mL in the mice. A score of 5 indicates normal health, while 0 indicates death.

00Figure 3. Bacteria present in 100µL of blood in each animal group after 96 h post infection.

The acquired data will undergo statistical analysis to determine if there is a significant difference in the amount of bacteria present in the animal groups after the treatments.
CITED LITERATURES
Abengaña et al. 2012. Comparative treatment of Pseudomonas aeruginosa burn wound infection
using bacteriophage MB08 and antibiotics. Retrieved from 20126077-81_PapaDMdC.pdf
Colindres et al.,. 2011. Comparing the activity of bacteriophages and antibiotics on Klebsiella
pneumoniae and Pseudomonas aeruginosa. Retrieved from 20115975-83_PapaDMdC.pdf
Dall, C. 2016. CDC: Resistant Salmonella causes 6,200 illnesses a year. Retrieved from
http://www.cidrap.umn.edu/news-perspective/2016/12/cdc-resistant-salmonella-causes-6200-illnesses-year
Hanif et al. 2015. Isolation and Antibiogram of Clostridium tetani from Clinically Diagnosed
Tetanus Patients. Retrieved from doi: 10.4269/ajtmh.15-0040
Islam ; Haneef. 2015. The Isolation and Bio-chemical Identification of Clostridium tetani.
Retrieved from The-Isolation-and-Bio-chemical-Identification-of-Clostridium-tetani.pdf
Lin et al. 2017. Phage therapy: An alternative to antibiotics in the age of multi-drug resistance.
Retrieved from doi: 10.4292/wjgpt.v8.i3.162
Nordqvist, C. September 2017. Everything you need to know about salmonella. Retrieved from
https://www.medicalnewstoday.com/articles/160942.php
Philippine Council for Health Research Development. 2014. Retrieved from
http://www.pchrd.dost.gov.ph/index.php/14-r-d-updates/3742-anti-microbial-resistant-salmonella-detected-in-commercial-swine
Public Health Agency of Canada. 2010. Retrieved from
https://www.msdsonline.com/resources/msds-resources/free-safety-data-sheet-index/salmonella-enterica-spp/
Raveendran R, Wattal C, Sharma A, Oberoi JK, Prasad KJ, Datta S. 2008. High level ciprofloxacin
resistance in Salmonella enterica isolated from blood. Retrieved from https://www.ncbi.nlm.nih.gov/pubmed/18227598
Schlecht, H ; Bruno, C. Januray 2015. Retrieved from
https://www.msdmanuals.com/professional/infectious-diseases/bacteria-and-antibacterial-drugs/fluoroquinolones
World Health Organization. January 2018. Antimicrobial resistance. Retrieved from
http://www.who.int/mediacentre/factsheets/fs194/en/
TIME TABLE

Activities Months
1st
2nd
3rd
4th
5th 6th 7th 8th 9th 10th
Writing of thesis proposal Oral presentation Preparation of materials for sample collection Sample collection Experimentation and laboratory analysis Drawing of conclusion Writing of first draft Writing of second draft Submission of final paper and manuscript Defense LINE ITEM BUDGET
Materials and Equipments 100, 000
Laboratory equipmentsReagents
Meat (swine) samples 5,000
Transportation2,000
Total: 107,000