Team:Carnegie Mellon/Project/Abstract

Abstract

Due to the widespread misuse and overuse of antibiotics, drug resistant bacteria now pose significant risks to health, agriculture and the environment. An alternative to conventional antibiotics is phage therapy. However, many temperate phage also form prophage. Our approach to antibiotic resistance is to engineer a temperate phage, Lambda, with light-activated production of superoxide. The fluorescent protein KillerRed was cloned into a plasmid vector and lambda gt11 with the IPTG inducible lac promoter. Lysogens were isolated and these strains were characterized and compared to E. coli with KillerRed from high-copy plasmids. Light activation of KillerRed resulted in decreased cell numbers. In addition, we modeled our system at multiple scales, including populations of phage and bacteria, KillerRed gene expression, ROS production, and effects of light. Having two methods of killing, lysis and superoxide, decreases the probability of developing resistance and our system overcomes the prior limitations of using wild-type temperate phages.

Impact

On September 16, 2013 the CDC released to the public “Antibiotic Resistance Threats in the United States, 2013”. This document is intended to raise public awareness of the problems associated with overuse and misuse of antibiotics and to outline the threats to society caused by these organisms. The organisms have been categorized by hazard level as urgent, serious and concerning. Over 2 million illnesses and 23,000 deaths per year are a direct result of antibiotic resistance. The CDC reports 4 major steps to tackle the problem including:

1. Preventing Infections, Preventing the Spread of Resistance
2. Tracking
3. Improving Antibiotic Prescribing/Stewardship
4. Developing New Drugs and Diagnostic Tests

Summary

There is a great need for new antimicrobial strategies. Phage therapy represents a completely different solution. Incorporating KillerRed phototoxicity provides another level of controlled killing. Phages are biological entities, which, theoretically, can be engineered to evolve at rates comparable of the natural evolution of bacteria if they happen to develop resistances to infection. Many phages are temperate, meaning that they can enter the lysogenic phase, which is undesirable for a killing phage. The addition of KillerRed to the system offers a second method of killing in the lysogenic phage. Thus, our system explores the possibility that temperate phages can also be used for phage therapy and bacteria killing applications. Our project establishes a first step in the production of phage therapies that

Background

Discovery of Antibiotics

Arsphenamine is an arsenic compound that was discovered in 1909, to have antisyphilitic properties by Sahachiro Hata and Paul Ehrlich. In 1929, Sir Alexander Fleming published the results of his study of a substance that he named penicillin. This compound was released by Penicillium fungi and killed bacteria. These antibiotics function by inhibiting an enzyme involved in crosslinking of the peptidoglycan cell wall of gram-positive bacteria. Resistance to the beta lactam antibiotics is derived from beta-lactamase, an enzyme that cleaves the drug and inactivates it. The gene for this enzyme is commonly used as a selectable marker in recombinant DNA laboratories to confer resistance to ampicillin. Today there are antibiotics of the cephalosporin family that are derived from the compound isolated in 1945 by Giuseppe Brotzu, from the fungus Cephalosporium acremonium.

The first synthetic antimicrobial compounds were the sulpha drugs, discovered in the early 1930’s. These drugs were developed at Bayer from dyes, they are bacteriostatic and act by inhibiting folate biosynthesis. Since the 1930’s thousands of modifications have been made to improve stability and functionality and decrease side effects. Where sulfonamides and sulfones are analogues of para-aminobenzoic acid, other medicines such as trimethoprim, methotrexate, pyrimethamine bind to dihydrofolate reductase and inhibit formation of tetrahydrofolic acid.

Aminoglycosides, tetracyclines and spectinomycin all inhibit protein synthesis by binding to the 30S ribosomal subunit. Chloramphenicol, lincomycin, clindamycin,Erythromycin bind to the 50S ribosomal subunit and fusidic acid binds to elongation factor G to inhibit protein synthesis. Inhibitors of nucleic acid synthesis include rifampin, rifamycin, rifampicin and quinolones such as nalidixic acid, ciprofloxacin, oxolinic acid.

Figure 1:Antibiotic resistance timeline since 1940

Figure 2: Antibiotic development pipeline since 1980

References

Antibiotic resistance threats in the United States, 2013. The Center for Disease Control. 9/16/2013.