Engineered Viruses Could Fight Drug Resistance

The surfacing of a variety of drugs for resisting antibiotic for disease-inflicting bacteria has already been a big issue and at the same a vital dilemma in treating all types of human diseases. This immediately requires another option, a substitute way of providing therapy to the human diseases. It has been found in previous researches and experiments the wonders of having bacteriophages highly considered to be “healing viruses. ” John MacGregor (2003) has brought up an intriguing issue regarding bacteriophages when he wrote his research article entitled “Set A Bug To Catch A Bug”.

Apart from the title, his words were “As the power of antibiotic wanes, viruses that hijack bacteria and smash them into pieces could be the answer to our prayers…”. He explained in his article the possibilities of replacing antibiotics as the solution to a lot of virus-causing diseases including virus infections. Bacteriophages were first discovered by a British chemist named E. H. Hankin. It was considered to be a virus in 1915 by Frederick Twort, a British bateriologisy. The occurrence of that first intrigue found by Dr. Hankin paved its way for more discoveries performed by a Canadian microbiologist named Felix d’Herelle. He agreed with Twort when he also considered it to be a virus and then later he named it as a “bacteriophage”. Upon his successful experimentations, he was confident that these bacteriophages will be very helpful and at the same time when he used them with the children who were almost dying dysentery at a hospital in Paris. The test solutions were distributed to every patient hoping that it will be effective, and fortunately, these cured the children for just one night.

With D’Herelle’s primary success, the use of phage therapy was further studied. From then on, the advantages it provides were widely spread globally. These page therapies are utilized in a variety of ways. It can be taken or given topically, orally, can be injected, using enemas and aerosols. Diseases that were treated by this phage therapy included urinary tract infections, typhoid and cholera. The use of phage therapy slowly faded when AMA or the American Medical Association reported contradictory results of using phages. Antibiotic age came in when penicillin was discovered by Alexander Fleming in 1982.

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Fleming’s discovery flourished for 20 years making the phage therapy out of sight. But still a lot of microbiologists became very attractive to what bacteriophages could provide medically, improving different aspects of health and curing more diseases. There was a time after the Communist era when phages were considered to be the “last resort” antibiotics when the decline for antibiotics took place. This was indeed an alarming situation worldwide. But this did not become hindrance in reviving the hidden attributes of bacteriophages.

Bacteriophages are defined by Toronto, Funke and Case (2001) as viruses that host in bacteria and in bacterial cultures, they can be grown easily. This has been significant since bacteriophages are the main sources of multiplying viruses. How do bacteriophages multiply? The basic procedure in which multiplication of viruses happens is just the same as the other viruses and it is not affected by any means by which the entering and the exiting of a virus into a host cell differs. Most life cycles of a virus are difficult to understand but a bacteriophage is an exception.

They are the easiest to be learned and be understood. Bacteriophages, also called “phages”, can be multiplied using two substitute processes. The first one is called the lytic cycle which leaves the host cell to a lysis or death. The second one is called the lysogenic cycle where the host cell lives. T-even bacteriophages such as T2, T4 and T6 are the phage types that are most studied. Using the bacterium Escherichia coli (E. coli) as a host, with the use of lytic cycle, multiplication of the T-even bacteriophages can be demonstrated easily from one process to another.

There are 5 stages involved starting from attachment, penetration, biosynthesis, maturation and release. During the first stage, attachment, the particles of the bactriophage and the bacteria collide. A chemical connection occurs between the attachment site from the virus and the bacterial cells’ complementary receptor site. A chemically produced interaction from the connection enables bonds that are weak to be formed from the two sites by using their fibered tail ends. During the stage of penetration, DNA is being injected by the T-even bacteriophages into the bacterium after connecting occurs.

This is done when an enzyme called the phage lysozyme is released by the tail of the bacteriophage which in turn destroys the walls of the bacterial cell. In the penetration process, the phage’s sheath tail contracts and the core of the tail enters the cell wall. If the core’s tip has already reached the plasma membrane, the DNA from the head of the bacteriophage will pass through the tail core, it will eventually enter the bacterial cell. During the stage of biosynthesis, the DNA of the bacteriophage will initiate synthesis direction of the components of the virus by the host cell.

Once the components of the virus are being put in place and are brought together into virions, the maturation stage occurs. When the lyses of the host cell and the new virions have already been released, it is referred to as the release stage (Toronto, Funke and Case; 2001). Bacteriophages have been found to exhibit a lot of features. They are tested to be of big use with local infections in relation to poor supply of blood like diabetic ulcers and infections of the bone. Unlike antiobiotics, these phages were keenly observed to multiply inside their host cell which enables them to penetrate more deeply to the area being infected.

Another distinguishing feature of phage therapies is their ability to inflict no allergies, resulting to a fewer side effects. Phage therapies in addition are easier and are cheaper to produce than antibiotics. On the other hand, bacteriophages have their limitations concerning their fatality once they have already killed the harmful bacteria. But these issues should not lower the hopes of the society depending on the future developments of phages. In an interview (Society for Gen. Micro. , 2008), they have shared that modern scientists and researchers have already found ways of prolonging the lifep of viruses.

This new and possible idea is by combining them chemically with polymers but still this is limiting since it will likely cause poisoning of the blood and is surely a big threat to one’s life. The main objective of this project is to determine the longetivity of the bacterium Escherichia coli’s gaining resistance ability in two different viral invaders: the bacteriophage T-4 and an antibiotic. At the end of this project, results should report a comparison between these two, answering which has the longest and the most effective invading mechanism.

Methods and procedures (Experiment protocol)

Throughout the following procedures of this project, a strict Aseptic Technique will be used. During the whole duration of the experiment, a strict technique called the Aseptic Technique will be applied. According to a web article research, written by Hauswirth and Sherk (2007), they defined the aseptic technique as an accumulation of unique practices acquired and a set of processes undergone whose conditions are carefully controlled with an objective of minimizing pathogen contamination. In any type of clinical setting, the technique is used to maximize and stabilize pathogenic organisms’ absence.

Its main goal is to simply protect a patient from infection and cease any possible spreading to other body parts. Prevention of infections are not achieved simply by sanitizing or disinfecting. In about more than 27 million surgical operations, surgical sites are the third most prone to more hospital infections prolonging, in effect, the hospital stay of the patient and at the same time, hospital bills become so expensive. The estimate was according to CDC or Center for Disease Control and Prevention (Hauswirth and Sherk, 2007).

The main objective of performing this experiment is to identify the required length of time for a common bacterium called Escherichia coli (E. coli) to achieve its maximum log phase growth. When these cells have already arrived in its expression of maximum amount of bacteriophage receptors, it will eventually lead to an immediate vulnerability to infection. The bacterium E. coli must always be utilized at this stage of growth all through out the procedures to be able to achieve desirable results upon comparing of two different experiments.

In beginning the procedures of the experiment, first, an overnight growth of culture will be prepared to be able to supply the growth curve with inoculum. The growth of the culture will be done in a shaking incubator with a temperature of 37 degrees Centigrade. The culture preparation will also be dependent on what culture is available, its slope, its colony or plate, and inoculate broth of the culture. Second, a 1 ml of culture that will be prepared overnight and a 99 ml of inoculate nutrient broth (NB) will be taken and will be placed in a flask that is sterilized and flat-bottomed type.

Through a process called resuspension, a sterilized tube containing a sample of 5 ml will be gently swirled, will be collected and will be marked Time Zero. The sterilized flat-bottomed flask will be placed inside the shaking incubator. Third, samples containing 5 ml each will be collected at a per hour interval. This will be done for 8 consecutive 8 hours and will be marked Time 1, Time 2, Time 3, Time 4 . . . Time 8. All samples will be stored at +4 degrees Centigrade. Fourth, the remains of the culture that has been prepared overnight will be left for one more night.

At exactly 9 am the next day, a last sample of the culture remains will be collected. Fifth, from a sample of 400 nm and another sample of 450 nm, the OD of each sample will be measured. The LB or NB will be used but will be left blank if necessary. If in case, the OD will exceed 1. 0, both sample 1 and sample 2 that were used in LB and NB will be diluted and will be read for the second time. Sixth, the strict aseptic technique will be used in preparing for colony counts in each sample. The amount of workable cells (per ml) will be identified.

Seventh, plotting will be done. A growth curve will be plotted and both the cell number and time will be involved in doing this. Eighth, another growth curve will be plotted. This time cell number and OD will be involved in the plotting. Ninth, the required time to reach the midway of the log phase growth will be identified. In doing this, cells within the time length, identified prior to the succeeding experiment, will be grown. Tenth and last step of these experiment procedures, the connection between the cells and the OD will be analyzed.

All results acquired all through out the process of this experiment will be recorded and will be evaluated accordingly.

References

1. Hauswirth, K. & Sherk, S. D. (2007) Aseptic Technique [Internet]. Available from < http://www. surgeryencyclopedia. com/A-Ce/Aseptic-Technique. html> [Accessed 8 May 2008]
2. Tortora, Funke & Case (2001) Microbiology: An Introduction. 7th ed. Addison-Wesley Longman, Inc. Craigie, J. (2002) The Significance and Applications of Bacteriophage in Bacteriological and Virus Research [Internet]. Available from <http://www. mmbr. asm. org> [Accessed 8 May 2008]

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