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NOVEL SOLUTIONS TO THE WORLD’S ANTIBIOTIC RESISTANCE CRISIS

Rishabh Seth (ris48@pitt.edu)

IS A POST-ANTIBIOTIC WORLD IMMINENT?

Approximately a century ago our world faced a major problem; bacteria were killing huge portions of the populus. Whether it was the common man or a general out at war, people were dying across the world and socioeconomic spectrum of these bacteria. Thankfully, Alexander Fleming managed to find a mold that could kill bacteria and inhibit their growth; penicillin. Suddenly the world was in a far better place. Militaries were not losing large portions of their soldiers to bacterial infection, and the infant mortality rate plummeted. Life expectancy rose significantly and all seemed to be well. However, now, 100 years later, we stand on the horizon of a world that is similar to that which existed before the discovery of penicillin. This is due to the adaptability of the bacteria we are combating. Bacteria are uniquely suited to quick evolution, with their short generations and ability to share DNA with other bacteria via conjugation. Since bacteria are able to temporarily fuse to share genetic information, resistance to antibiotics can be shared between bacteria of the same generation without the necessity to reproduce. Resistance to antibiotics, of course, is the product of random mutation. However, with the sheer number of bacteria there are and their ability to share this resistance, antibiotics tend to become quickly ineffective after they are released into the ecosystem. This was not a problem until relatively recently because our broad spectrum of antibiotics could kill practically any bacteria. In the past few years, however, it has become exceedingly clear that we are at a point where the bacteria’s adaption to our antibiotics has outpaced our production of new medicines. Our survival demands a new, novel set of antibiotics that rely on mechanisms that previous antibiotics have not, so the bacteria are less likely to be able to react to them. These novel antibiotics may just be chlorinated plant proteins, which have recently shown themselves as a promising solution to our problem of a post-antibiotic planet.

CHLORINATED PLANT PROTEINS

The reason why we, as a society, find ourselves in the predicament of antibiotic resistance is that our antibiotics have largely worked in similar ways over the last century. Most of the antibiotics found in the 20th century worked by inhibiting a bacteria’s ability to synthesize a cell wall. While this was largely an effective way to kill bacteria, as it caused the bacteria to rupture and die when they attempted replication, now that bacteria have adapted to resist antibiotics that function in this way, we find ourselves with limited options. These antibiotics that functioned in similar ways were made useless by bacterial evolution almost on release.

Figure 1 [1]Timeline of Antibiotic Introduction and Resistance

For a new set of antibiotics to be effective in the long term, they must kill bacteria in a fundamentally different way. Thankfully, the new wave of antibiotics currently under research, especially those that involve the use of chlorinated plant proteins, do exactly that. There are three antibiotics

University of Pittsburgh, Swanson School of EngineeringSubmission Date 10.31.2017

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that show unique mechanisms in dealing with pathogens: intragenic antimicrobial peptides, chlorinated emodin, and diphenyleneiodonium chloride that have also had research published in Nature magazine, one of the premier scientific journals.

METHODS OF FUNCTION

The methods of function of each of these antibiotics differs significantly, despite the similarity of the sources of the antibiotics. Intragenic antimicrobial peptides, or IAP’s, are the most varied group of antibiotics, as the other two examples are individual chemicals. Out of the 21 IAP’s there are many that work on some of the pathogens tested but not others. Specifically, “Tc02 inhibited the growth of yeasts and filamentous fungi more efficiently than Gram-positive and -negative bacteria, while Tc08 presented an opposite pattern of activity,” and it seems that most of the IAP’s fall into one of these general categories [2]. At04 fell into the category of Tc08, whereas Tc06, At01, Cs01 and Zm01 fall into the other category. There is also a third category that includes the IAP’s that are generally effective, such as At02, Gr01 and Gr02. To go along with the general usefulness of these IAP’s as antimicrobial or anti-starch agents, these proteins do not negatively impact the human body. This is partially because many of these proteins are microbistatic, not microbicidal meaning that, while they inhibit growth, they do not kill the microbes they are targeting. This allows for the internal microbiome of the human body to remain relatively unperturbed. About our blood specifically, Tc02, Gr01 and Gr02 all do cause blood cells to lyse, but at far higher levels than what would be needed for fungicidal or antibacterial use. As a demonstration of the fungicidal properties of these IAP’s, Tc02 inhibited the growth of basidiospores by 95% at a concentration of 16 µM. One of the papers that was the source for this paper on the 21 IAP’s showed IAP’s being effective at even lower concentrations, “They [the IAP’s] both inhibited the in vitro growth of X. axonopodis pv. glycines, the causative agent of the bacterial pustule disease, at 5 and 10 µM, respectively,” showing how the IAP’s are useful at concentrations like those of current antibiotics [3].

The second main paper on new antibiotics is on chlorinated emodin, or CE, and its use as a widely applicable antibiotic. This paper is the one that best explains the mechanism of its antibiotic and shows a singular antibiotic that functions effectively against many microbes, and so this is the best looking of the three sets of antibiotics I am covering here. Emodin, which is a chemical found in rhubarb, is itself known to inhibit microbial growth, but when it is chlorinated its antimicrobial properties grow significantly. Details on the types of bacteria that CE works best on are also outlined, as it seems that CE is more effective on gram-positive bacteria, or

bacteria that have a thick cell wall made of peptidoglycan, than on gram-negative bacteria, which have a thick plasma membrane instead. While this may initially seem to limit the usefulness of CE, as it doesn’t seem to work on gram-negative bacteria, “CE completely inhibited the growth of all 18 strains of clinically isolated Bacteroides fragilis;” a gram-negative bacterium [4]. This proved to the researchers that there was clearly a way for CE to be effective on gram-negative bacteria, and that way is to destabilize the outer membrane of the bacteria. CE worked on B. fragilis because it has a strangely composed outer membrane, or OM, and by disrupting the OM the researchers made even the gram-negative bacteria vulnerable to CE. What is the most interesting about CE, however, is how it kills bacteria in multiple, novel ways. These include increasing the permeability of the bacterial membrane, forcing leakage of potassium from the bacterium, and depolarization of the bacterial cell membrane. All three of these approaches differ from that of penicillin, which simply inhibited the replication of bacteria until it ruptured. Increasing the permeability of the bacterial cell membrane allows for chemicals toxic to the bacteria to get in, resulting in cell death. The forced leakage of potassium also shows damage to the bacterial cell membrane. Finally, depolarization of the cell membrane allows for certain ions that normally could not enter the bacteria to do so, also possibly leading to its death. Another interesting effect CE has on bacteria is compressing their DNA via an electrostatic interaction, but this does not directly lead to bacterial death. Additionally, CE was tested on hamster lung cells and proved to not cause any damage to the cells. This implies that it is safe for further testing and may potentially be safe for use in human medicine. Something that is rather interesting is that one of the sources of this paper mentions how emodin can find its roots in Chinese Ayurveda, “What is more, Phylloquinone B (16) and C (17) [along with emodin] were also isolated from the leaves of Polygonum cuspidatum [a plant used in traditional Chinese medicine],” showing the potential antibiotic properties of traditional medicine [5].

While CE certainly appears to be the most promising future antibiotics, diphenyleneiodonium chloride, or DPIC, is also another viable option. As another chloride, DPIC bears some similarities to CE. The most significant is that it, like CE, is not very effective against gram-negative bacteria on its own. Something that differentiates DPIC from CE is that it can, “potentially drastically reduce the evolution of drug resistance bacteria among the NRP population that otherwise could spontaneously become refractory to one or more drugs,” in combination with PZA [6]. If this is true, then DCIP is unique in its function and should be the prospective antibiotic with the most resources poured into it. Another significant feature of DPIC is its ability to function inside bone marrow, a place where many bacteria multiply. DPIC’s ability to both kill bacteria and inhibit bacterial growth allows it to be used to prevent a “cured” infection from relapsing, a trait that not all

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antibiotics have. A final trait DPIC has is its compatibility with tuberculosis drugs, which means that it is even more widely applicable than previously stated. All three of these antibiotics have their own uses, and give me hope for the future. Antibiotic resistance is important as a problem for engineering, and me, to solve.

IMPORTANCE OF NEW ANTIBIOTICS

The importance of developing new antibiotics to society is self-evident; were we to not do so our society would revert to the state it was in over a century ago. For a more statistical view of the sociological and economic damage, “Recent modelling by the Independent Review on AMR, chaired by Lord O'Neill, has predicted that AMR will cause an additional 10 million deaths per year and a loss of up to US$100 trillion from global GDP by 2050;” a definite cause for worry [6].

Figure 2 [7]Deaths Because of Antimicrobial Resistance

Something worth noting here that this is just over the next third of a century, and as the bacteria get a larger and larger lead on us it will only get worse, unless we develop new antibiotics. This should be seen as an engineering problem as engineering is essentially all about solving the problems society has. Constructing a novel solution to a problem is what engineering is all about, so I find antibiotic development to clearly be bioengineering. Of course, I would not have chosen this topic were it not close to my heart. I am personally hoping to go into antibiotic R&D because not only do I want to solve what I see as possibly the greatest threat to humanity, but also because my grandfather was infected with antibiotic resistant bacteria three years ago and nearly died before being saved by an antibiotic that was kept in reserve. That is why the development of new antibiotics is significant to both me, engineering, and society as a whole and why this research is so important for the future of our society and gives me hope.

SOURCES

[1] C. Lee Ventola. “The Antibiotic Resistance Crisis.” NIH. 4.2015. Accessed 10.31.2017

[2] M. H. S. Ramada, G. D. Brand, F. Y. Abrão, M. Oliveira, J. L. Cardozo Filho, R. Galbieri, K. P. Gramacho, M. V. Prates, C. Bloch Jr. “Encrypted Antimicrobial Peptides from Plant Proteins.” Nature. 10.16.2017. Accessed 10.31.2017[3] Guilherme D. Brand, Mariana T. Q. Magalhães, Maria L. P. Tinoco, Francisco J. L. Aragão, Jacques Nicoli, Sharon M. Kelly, Alan Cooper, Carlos Bloch Jr. “Probing Protein Sequences as Sources for Encrypted Antimicrobial Peptides.” Plos. 9.28.2012. Accessed 10.31.2017[4] Feixia Duan, Guang Xin, Hai Niu, Wen Huang. “Chlorinated emodin as a natural antibacterial agent against drug-resistant bacteria through dual influence on bacterial cell membranes and DNA.” Nature. 10.05.2017. Accessed 10.31.2017[5] WeiPeng, RongxinQin, XiaoliLi, HongZhou. “Botany, Phytochemistry, Pharmocology, and potential application of polygonum cuspidatum.” ScienceDirect. 7.05.2013. Accessed 10.31.2017[6] Manitosh Pandey, Alok Kumar Singh, Ritesh Thakare, Sakshi Talwar, Pratiksha Karaulia, Arunava Dasgupta, Sidharth Chopra, Amit Kumar Pandey. “Diphenyleneiodonium Chloride (DPIC) displays broad-spectrum bactericidal activity.” Nature. 9.14.2017. Accessed 10.31.2017[7] Rebecca Sugden, Ruth Kelly, Sally Davies. “Combatting antimicrobial resistance globally.” Nature. 2017. Accessed 10.31.2017

ACKNOWLEDGEMENTS

I would like to thank my family, for providing an environment in which I grew to love science and teaching me to love helping people. I thank my fellow engineers on Nordenburg floor 6 for helping me write this paper. I also thank the speakers at the career conference, for taking their time to show us a little bit about how it is to be a professional engineer. Finally, I thank the University, for providing me the resources to pursue my dream.

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