1 April 15, 2012 Writing center

Bacterial Phage Therapy Bacterial phage therapy is the use of bacteriophages to treat pathogenic bacterial infections. Bacteriophages are special viruses that are very specific in the bacteria that they can kill. This is important because the uses of phages hypothetically are so specific that they wont harm the host. The use of phages are becoming more important within society since many antibiotics are so strong they do more harm than good. Bacterial phage therapy was founded in 1915 by British bacteriologist Frederick W. Twort, who found the small organism that can infect bacterial cells and in turn kill them from its infection. After he found this small virus. Twort decided not to pursue any more research about it and left it for some other scientist to discover. Felix d'Hrelle in September 3, 1917, was able to isolate the bacterial phage without any previous knowledge of Frederick W. Tworts research which happened two years previously. In August 1919 d'Hrelle successful use of phages on humans was administered, he was able to cure dysentery, which is a bacterial infection of intestine and colon. In 1923 George Eliava, an associate of d'Herelle, was able to found Eliava Institute in Georgia. This institute has been one of the leaders in phage therapys research since its founding. All these findings of using bacterial phages to heal infections led to a massive pharmaceutical race between all of the major world powers; this lead many major corporations to invest large amounts of money on banks on phage therapy, which was considered the new pharmaceutical future. Unfortunately, in 1939 the discovery of penicillin and the major production of antibiotics because of World War Two caused the Western world to forget about phage therapy. However, with the

2 blockade of information about antibiotics to the eastern world, the USSR and Germany still researched much about phage therapy for their troops to prevent bacterial infection. The end of the Second World War and the end of the Cold War truly hurt phage therapy research because of the spread of antibiotic information. Fortunately, For 80 years Georgian doctors have been treating local people, including babies and newborns, with phages. As a result of the development of antibiotic resistance since the 1950s and an advancement of scientific knowledge, there has been renewed interest worldwide in the ability of phage therapy to eradicate bacterial infections and chronic polymicrobial biofilm, along with other strategies. (Brussow 2005) The Georgian doctors have been keeping phage therapy alive ever since 1923 and this has been very influential now during our time where more and more bacteria are gaining resistance to our antibiotics. Now with dangerous and controversial antibiotics being used more and more the need for a safer way to take care of these super strains of bacteria are necessary. The main types of bacteriophage are used and research either for human or agricultural reasons are Enterobacteria phage T4/T5/T7. The way the bacteriophage affects the bacteria is during replication which can be defined in four steps. Attachment, integration, synthesis, and release these steps all happen in the to bacteriophage life cycles. Bacteriophages have a lytic cycle or a lysogenic cycle; some of the phages may even have both cycles if needed for its environment. The first cycle is the lytic cycle: bacterial cells are broken open and destroyed after immediate replication of the virion. As soon as the cell is destroyed, the phage progeny can find new hosts to infect. Lytic phages are more suitable for phage therapy. The second cycle is lysogenic pathway: where the viral dna is integrated into the host but doesnt go through the lytic path since conditions arent favorable (Brussow 2005).

3 These pathways and knowing how the phage interacts with the cell we can now determine how to effectively infect bacteria.The two major infections that I will be talking about are Escherichia coli in acute diarrhea in children and E Coli infections within commercial cow farms: The World Health Organization estimates that 5 million children die each year as a consequence of acute diarrhoea(Snyder & Merson, 1982). Escherichia coli is the cause of a third of cases of childhood diarrhoea in developing and threshold countries (Albert et al., 1995) and is also the most prominent cause of diarrhoea in travelers to developing countries (Black, 1990). Due to its malleable genetic character, E. coli has one of the widest spectra of disease of any bacterial species (Donnenberg,2002). The recent emergence of E. coli O157 as a major food pathogen is a lively reminder of its dynamic character. Furthermore, Shigella species, the cause of dysentery, taxonomically constitute a subspecies of E. coli. In addition, effective treatment and prevention measures are lacking for E. coli diarrhoea. The mainstay of treatment is oral rehydration (Bhan et al., 1994). This simple and inexpensive measure has saved countless lives, but it does Not influence the natural course of disease and has no intrinsic antibacterial activity. (Brussow et.all,2005) The World Health organization was able to determine that the majority of all acute diarrhea deaths of children had been as a result of an E.Coli infection in the bowls. From the research they were able to see many things about the viability of phage therapy for acute diarrhea. The experiments that were held to regulate how well phage cocktails worked on humans with diarrhea used many different strains of phages that were isolated. This was done first by isolating two major subcategories of E.Coli phages, lambda like Siphoviridae that were isolated from healthy stool in

4 hospitals and T4-like Myoviridae in children who had acute diarrhea. These two major groups of E coli helped give the cocktail a larger range host range meaning it could affect more different strains of E coli in a persons system therefor helping them get over the diarrhea faster. The results were astonishing, Phage administration was associated with a 3 to 8-fold decrease in dysentery incidence (1to8 vs 6to7 episodes per 1000 children from treatment and placebo group, respectively). The culture-confirmed incidence was decreased 2?6-fold by phage application (0?7 vs 1?8 episodes, respectively). Phage exposure also decreased the incidence of any form of diarrhoea (15 vs 45 episodes per 1000 children 6 to 12 months old in treatment and placebo group, respectively). This latter observation suggests a protective effect of the anti-Shigella phage preparation against other serotypes of E. coli. Protective effects were most pronounced in children younger than 3 years. (Brussow,2005) Even with these field changing results there were three major problems that the researches had to overcome either back when phages were discovered or during the trials practicality, safety, and clinical versus actual. The Practicality of phage therapy was first addressed back during the time of Felix d'Hrelle. The issue was in what form the phage therapy should be administrated in either a liquid or in a lyophilized form (freeze-dried powder) and how these forms will affect the medicines effectiveness. In the end the lyophilized from was found to be superior. The reason for this is that it could be stored in massive quantities in a relatively low amount of volume, the shelf life was stable in a room temperature room for about fourteen months without losing it potency, and with the freeze-dried from it could be given orally instead of intravenously (through veins). The only down

5 side to this form is that in extreme temperatures the phages became too acidic and became less effective. Even to this day the oral applications of bacteriophages are used. Even though some techniques have changed such as using gel casuals instead of lyophilized pills. In these gels capsule pills the bacteriophages are repressed from activation until they are introduced to the GI tract. The next concern that need to be address was that of the mutation of the phage itself. When phage therapy first was performed, many researchers did not know that there were many different type of phages that just like their bacterial counterparts can adapt with time to counteract each other. The major players in E coli bacteriophages would be T4/T7/T2 strains. When these bacteria would mutate and change their binding sites to counter act the phage. The specific phages that effect that bacteria would also go through an alteration themselves and enlarging the host range which made them lose some of its specificity so it could infect the bacteria. Meaning when bacterial host changes some of the phages will change with it to attack the cell. The last and most crucial aspect of phage therapy that researchers had to deal with was safety. The reason that there is so much concern about phage therapy is that when one uses this bacterial virus it tends to stay within the body for several years. This gives the person added protection, but also could give the body problems just in case it mutates. The years of research had shown that these genes didnt mutate with formation of any toxic genes that could harm us. The phages were found to be even better than most antibiotics that kill and harm your body organs. Also it is extremely cost effective if you look at from an economic point of view. This is because Bactria can replicate extremely fast and make many phages when harvested. All of this research being done shows that phage therapy is safe for humans, but still there are not many companies or countries using them against human infection because of the chance of

6 mutation. Though we do not like to use it on ourselves we will use them on animals we eat such as cows. The use of phages on cows led to the same results as they did on humans except for two exceptions. Researchers found that the use of low dosages of phages at the first sign of diarrhea was the best way of action since the phages were able to reproduce exponentially in 30 minutes about ten to the ten of what was put in. In comparison where high dosage did the something but it not administer in the right time frame it was ineffective. The second difference the cows had in comparison to the humans is that: Calves held in a room previously occupied by phage exposed calves could no longer be infected with the entero-pathogen, coming close to dHerelless initial idea of infectious protection by phages(Brussow,2005). This shows that instead of bacteria gaining resistance to the antibiotic, which normally happens, the cow gains resistance to the bacteria from the phage. The use of bacteriophage therapy is becoming more and more prevalent within our society. Many people do not even know that many of our vegetables, cheese, and other food products are already treated with bacteriophages. In August, 2006 the FDA approved LMP-102 as a food additive to target and kill Listeria monocytogenes. LMP-102 was approved for treating RTE poultry and meat products. In October of that year the FDA approved a product by EBI using bacteriophages on cheese to kill the Listeria monocytogenes bacteria, giving them GRAS status (Generally Recognized As Safe). In July 2007, the same bacteriophages were approved for use on all food products (FDA et.all 2008). The future that bacteriophage therapy has in store for us could be limitless. With the use of stronger and stronger antibiotics on the rise, the need for a different means of preventing or dealing with infections is need. Also with the added benefit of being treated with phages, the body can obtain resistance to many strains of E coli and other bacteria. This could mean that antibiotics would be useless. Even though phage therapy may be

7 used more and more our childrens children will come to the same problem that phage therapy and antibiotics are not enough. But we have a long way to go before that happens because the only place in the world where phage therapy on humans is legal is in Georgia. Soon the world will have to realize that phage therapy is actually a good means to kill bacterial infections rather than using harmful antibiotics ( Brussow 2005).

Referance Brussow, H. "Phage Therapy: the Escherichia Coli Experience." Microbiology 151.7 (2005): 2133-140. Print.

8 USA. FDA. FDA GRAS. Web. <http://google2.fda.gov/search? q=phage+page&spell=1&client=FDAgov&lr=&proxystylesheet=FDAgov&output=xml_no_dtd&i e=UTF-8&site=FDAgov&access=p>. Zuber, S., C. Ngom-Bru, C. Barretto, A. Bruttin, H. Brussow, and E. Denou. "Genome Analysis of Phage JS98 Defines a Fourth Major Subgroup of T4-Like Phages in Escherichia Coli." Journal of Bacteriology 189.22 (2007): 8206-214. Print.

Abstracts
The specter of antibiotic-resistant bacteria has provoked renewed interest in the possible use of bacteriophages to control bacterial infections. We argue that clinical application of phage therapy has been held back by a failure to appreciate the extent to which the pharmacokinetics of self-replicating agents differ from those of normal drugs. For self-replicating pharmaceutical agents, treatment outcome depends critically on various density-dependent thresholds, often with apparently paradoxical consequences. An ability to predict these thresholds and associated critical time points is a necessity if phage therapy is to become clinically practicable. (Clin Pharmacol Ther 2000;68:225-30.) The relationship between endemic bacteriophages infecting E. coli O157:H7 (referred to as phage) and levels of shedding of E. coli O157:H7 by cattle was investigated in two commercial feedlots in southern Alberta, Canada. Between May and November 2007, 10 pens of cattle were monitored by collection of pooled fecal pats, water with sediment from troughs, manure slurry from the pen floor, and rectal fecal samples from individual animals (20 per pen) at two separate times. Bacteriophages infecting E. coli O157:H7 were detected more frequently (P < 0.001) after 18 to 20 h enrichment than by initial screening and were recovered in 239 of 855 samples (26.5% of 411 pooled fecal pats, 23.8% of 320 fecal grab samples, 21.8% of 87 water trough samples, and 94.6% of 37 pen floor slurry samples). Overall, prevalence of phage was highest (P < 0.001) in slurry. Recovery of phage from pooled fecal pats was highest (P < 0.05) in May. Overall recovery did not differ (P > 0.10) between fecal grab samples and pooled fecal pats. A higher prevalence of phage in fecal pats or water trough samples was associated (P < 0.01) with reduced prevalence of E. coli O157:H7 in rectal fecal samples. There was a weak but significant negative correlation between isolation of phage and E. coli O157:H7 in fecal grab samples (r _ _0.11; P < 0.05). These data demonstrate that the prevalence of phage fluctuates in a manner similar to that described for E. coli O157:H7. Phage were more prevalent in manure slurry than other environmental sources. The likelihood of fecal shedding of E. coli O157:H7 was reduced if cattle in the pen harbored phage. DOI 10.1128/AEM.02100-08.

Phages have been proposed as natural antimicrobial agents to fight bacterial infections in humans, in animals or in crops of agricultural importance. Phages have also been discussed as hygiene measures in food production facilities and hospitals. These proposals have a long history, but are currently going through a kind of renaissance as documented by a spate of recent reviews. This review discusses the potential of phage therapy with a specific example, namely Escherichia coli. DOI 10.1099/mic.0.27849-0