The Triple Helix @ UChicago

Spring 2016

"Staying Ahead of Infection: A New Plan of Attack" by Irena Feng

 

Before the advent of antibiotics, common infections 8killed quickly, as there were no drugs to stop them. When antibiotics were first developed in the early 20th century, they were seen as miracle cures; the antibiotics eliminated the bacteria easily by killing them or interfering with their growth enough to allow the body’s immune system to destroy them. However, almost a century after the first antibiotic was discovered, we may be heading back into a new post-antibiotic era as bacteria begin to find new ways to fight back.

The Superbug

Antibiotic resistance refers to when an antibiotic is no longer effective against its target, often because the bacteria have changed to reduce their vulnerability to the drug. The development of resistance against certain drugs was inevitable; just as natural selection favors survival of the fittest, so does our use of antibiotics, which favors the few resistant bacteria, allowing them to outlive their vulnerable peers and multiply into resistant strains, colloquially known as superbugs. For years, the scientific community’s response to these emerging strains of resistant bacteria was new antibiotics; however, the speed at which resistance can spread creates new challenges. 

Bacteria use horizontal gene transfer methods such as conjugation to physically share resistance genes. First discovered in 1946[1], conjugation is the bacterial version of sexual recombination between cells with or without a DNA sequence called the F plasmid (F+ and F- cells, respectively), spreading the F plasmid throughout a population of bacteria. 

The Hfr strain of bacteria takes this one step further. In the Hfr strain, as the F plasmid is transferred from donor to recipient bacterium, it is not only taken up in the recipient bacterium but also integrated into its chromosome[2], becoming a permanent portion of the recipient’s genome and resulting in a high frequency of genetic recombination. When conjugation occurs again after that, replication and transfer of the F plasmid inadvertently brings along neighboring genes in the donor cell. In this way, conjugation can lead to not only the transfer of F plasmid but also of additional genetic material. This allows genes that provide antibiotic resistance to be transferred between bacteria, quickly making an entire population of bacteria resistant rather than keeping the resistance to a single bacterium. 

The World Health Organization reported in 2014 that antibiotic resistance was a serious threat to public health[3] worldwide when it surveyed over 100 countries and found that in many cases, antibiotics against common infections like pneumonia and sepsis no longer work in the patients that need them. In some countries, over half of people treated could not use the antibiotics available because the antibiotic-resistant bacteria rendered available antibiotics useless. An even more severe consequence of antibiotic resistance is increased mortality rates. For example, staph infections are caused by staphylococcus bacteria, but can usually be treated with a widely-used class of antibiotics called beta-lactam antibiotics[4], which include penicillin and methicillin. However, people infected with MRSA (methicillin-resistant Staphylococcus aureus) are 64% more likely to die when compared to those infected with a non-resistant form[5], demonstrating the dangers inherent in multidrug-resistant bacteria like MRSA.

How Phage Therapy Works

A new plan of attack against these multi-drug resistant bacteria involves a new weapon: viruses. While antibiotics target multiple species of bacteria (sometimes even off-targeting our natural helpful bacteria), viruses that target bacteria – called bacteriophages – focus on specific species of bacteria, and sometimes even specific strains within a species[6]. A bacteriophage works against bacteria by injecting its own DNA into the bacteria, essentially hijacking the bacteria’s replication mechanisms to replicate itself and produce more bacteriophages. The propagation of this cycle depends on the availability of the target[7]; when there no more bacteria that match the bacteriophage, the phage ceases to infect and kill. 

One concern about using bacteriophages is the lysing process. In lytic phages, the bacteria are taken over and forced to produce phages until the cells explodes, killing the cells and releasing phages into the body system to infect more bacteria. However, when the bacteria are destroyed, their other contents are also spilled into the surroundings. These contents contain proteins and toxins that could bring further complications and cause the body to overreact to dying bacteria[8]. Scientists at MIT[9] are working to counter this by producing phages that kill the bacteria without lysing them. In these phages, nicknamed phagemids, there are small pieces of DNA instead of a full-fledged viral genome. Phagemids kill the bacteria but also prevent lysis, thus keeping the bacterial toxins within the bacteria. The dead bacteria can then be eliminated by the body’s natural defenses. 

Phage therapy can also be used to enhance the effectiveness of regular antibiotics, as phages can be used to sensitize the bacteria to the antibiotics by removing the genes conferring antibiotic resistance. At Tel Aviv University[10], researchers used bacteriophages to insert into bacteria a gene-editing system called CRISPR/Cas9 to destroy the genes responsible for resistance. The engineered phage contains CRISPR genes with short interjections that are expressed as RNA sequences designed to target specific DNA sequences. These RNA sequences then associate with Cas9 (an RNA-guided nuclease) which cuts the target DNA as directed by the RNA sequences, removing and destroying the targeted DNA sequence. Using genetic engineering, we can manufacture specific sequences that can target whatever we want, including antibiotic-resistance genes. One potential concern is that such a method would require prior knowledge of the specific sequences conferring resistance, but this concern is currently being addressed by experimenting with DNA sequencing that could make it easier to target and excise specific sequences. With the ability to resist antibiotics gone, the targeted bacteria may once again be vulnerable to the antibiotics we have available.

Viral Revival

With an increasingly better understanding of the mechanisms of action and the relative safety profiles of different treatments, phage therapy as a clinical option is becoming increasingly popular. In the early 2000s, clinical trials began to analyze immune responses to phages[11]; in 2015, clinical trials began to test for the safety dosages for phage cocktails (multiple phages combined) and other phage components, from specific enzymes to phage tail proteins. The timeframe for bacteriophage development is also much shorter; while antibiotic development could take years, selecting new phages against bacteria is a relatively short process that could take at most a few weeks[12]. 

The investigation of phage therapy against bacteria began around the same time as antibiotics hit the markets, and eventually yielded to antibiotics since the antibiotics were easier to produce and standardize[12]. However, as we increasingly come face-to-face with resistant bacteria nowadays, it may be time to reconsider phage therapy’s consignment to the past.

References

[1] Griffiths, Anthony JK, William M. Gelbart, Jeffrey H. Miller, and Richard C. Lewontin. 1999. “Bacterial Conjugation.” In Modern Genetic Analysis. New York: W. H. Freeman. http://www.ncbi.nlm.nih.gov/books/NBK21351/. 
[2] Griffiths, Anthony JF, Jeffrey H. Miller, David T. Suzuki, Richard C. Lewontin, and William M. Gelbart. 2000. “Bacterial Conjugation.” In An Introduction to Genetic Analysis, 7th ed. New York: W. H. Freeman. http://www.ncbi.nlm.nih.gov/books/NBK21942/. 
[3] “Antimicrobial Resistance: Global Report on Surveillance 2014.” 2014. World Health Organization. http://apps.who.int/iris/bitstream/10665/112642/1/9789241564748_eng.pdf? ua=1. 
[4] Elander, RP. 2003. “Industrial Production of B-Lactam Antibiotics.” Applied Microbiology and Biotechnology 61 (5): 385–92. doi:10.1007/s00253-003-1274-y. 
[5] World Health Organization. 2014. “WHO’s First Global Report on Antibiotic Resistance Reveals Serious, Worldwide Threat to Public Health.” WHO Media Centre. April 30. http://www.who.int/mediacentre/news/releases/2014/amr-report/en/. 
[6] Holmfeldt, Karin, Mathias Middelboe, Ole Nybroe, and Lasse Riemann. 2007. “Large Variabilities in Host Strain Susceptibility and Phage Host Range Govern Interactions between Lytic Marine Phages and Their Flavobacterium Hosts.” Applied and Environmental Microbiology 73 (21): 6730–39. doi:10.1128/AEM.01399-07. 
[7] Borysowski, Jan, Ryszard Miedzybrodzki, and Andrzej Gorski, eds. 2014. Phage Therapy: Current Research and Applications. Caister Academic Press. http://www.horizonpress.com/phagetherapy. 
[8] Staropoli, Nicholas. 2015. “Swan Song for Antibiotics? Can Phage Therapy and Gene Editing Fill the Gap?” Genetic Literacy Project. July 26. https://www.geneticliteracyproject.org/2015/07/26/swan-song-for-antibiotics-can-phage-therapy-and-gene-editing-fill-the-gap/. 
[9] Krom, Russell J., Prerna Bhargava, Michael A. Lobritz, and James J. Collins. 2015. “Engineered Phagemids for Nonlytic, Targeted Antibacterial Therapies.” Nano Letters 15 (7): 4808–13. doi:10.1021/acs.nanolett.5b01943. 
[10] Yosef, Ido, Miriam Manor, Ruth Kiro, and Udi Qimron. 2015. “Temperate and Lytic Bacteriophages Programmed to Sensitize and Kill Antibiotic-Resistant Bacteria.” PNAS 112 (23): 7267–72. doi:10.1073/pnas.1500107112. 
[11] Miedzybrodzki, Ryszard, Jan Borysowski, Beata Weber-Dabrowska, Wojciech Fortuna, Slawomir Letkiewicz, Krzysztof Szufnarowski, Zdzislaw Pawelczyk, et al. 2012. “Clinical Aspects of Phage Therapy.” In Advances in Virus Research, 83:73–121. Elsevier, Inc. http://www.sciencedirect.com/science/article/pii/B9780123944382000037. 
[12] Madhusoodanan, Jyoti. 2016. “Viral Soldiers.” The Scientist, January 1. http://www.the-scientist.com/?articles.view/articleNo/44785/title/Viral-Soldiers/. 
[13] Sulakvelidze, Alexander, Zemphira Alavidze, and J. Glenn Morris, Jr. 2001. “Bacteriophage Therapy.” Antimicrobial Agents and Chemotherapy 45 (3): 649–59. doi:10.1128/AAC.45.3.649-659.2001. 

 
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